JP2004531235A - STREPTOCOCCUSPYOGENES polypeptides and corresponding DNA fragments - Google Patents

Abstract

本発明は抗原、より特にStreptococcus pyogenes（またグループＡStreptococcus(GASと呼ばれる）細菌性病原体の抗原であって予防、治療および／または診断のためのワクチン成分として有用であるものに関する。Embedded image

The present invention relates to antigens, more particularly antigens of the bacterial pathogen Streptococcus pyogenes (also called Group A Streptococcus (GAS)), which are useful as vaccine components for prevention, treatment and / or diagnosis.

Description

好ましい置換はまた、対応するＬ−アミノ酸についてのＤ−エナンチオマーの置換を含む。
【００４０】
代替的アプローチでは、本発明のポリペプチドのアナログは、図３に開示の置換を含む。
代替的アプローチでは、アナログは、例えば、望まれるポリペプチドに効率的にタグを付加することによる、精製をより容易にさせる部分を含む、融合タンパク質であり得る。それは「タグ」を除去することが必要であり得、またはそれは融合ポリペプチドが有用であるのに十分な抗原性をそれ自体保持する場合であり得る。
【００４１】
相同性のパーセンテージは、アミノ酸型の、同一性のパーセンテージ加えて類似性または保存のパーセンテージとして定義される。
【００４２】
ある実施態様では、本発明のアナログまたはポリペプチドは、図に示されるこれらの配列またはそのフラグメントと約７０％同一性を有する。すなわち、残基の７０％は同じである。さらなる実施態様では、ポリペプチドは、７５％より大きい相同性を有する。さらなる実施態様では、ポリペプチドは８０％より大きい相同性を有する。さらなる実施態様では、ポリペプチドは、８５％より大きい相同性を有する。さらなる実施態様では、ポリペプチドは、９０％より大きい相同性を有する。さらなる実施態様では、ポリペプチドは、９５％より大きい相同性を有する。さらなる実施態様では、ポリペプチドは、９９％より大きい相同性を有する。さらなる実施態様では、本発明のポリペプチドのアナログは、約２０より少ないアミノ酸残基置換、修飾または欠失を有し、そしてより好ましくは１０より少ない。
【００４３】
プログラム、例えばＣＬＵＳＴＡＬプログラムを使用し、アミノ酸配列を比較することができる。このプログラムは、アミノ酸配列を比較し、そして適当なようにいずれかの配列中にスペースを挿入することによって至適アラインメントを見出す。至適アラインメントのためにアミノ酸同一性または類似性(アミノ酸型の、同一性加えて保存）を計算することが可能である。ＢＬＡＳＴｘのようなプログラムは、類似配列の最長のストレッチをアラインメントし、そしてフィットに値を割り当てる。こうして、類似性のいくつかの領域が見出される比較であって、それぞれが異なるスコアを有するものを取得することが可能である。同一性分析の両方の型を本発明で企図する。
【００４４】
代替的アプローチでは、アナログまたは誘導体は、例えば望まれるタンパク質またはポリペプチドに効率的にタグ付加することによって、精製をより容易にさせる部分を取りこませる融合ポリペプチドであり得、「タグ」を除去することが必要であり得、またはそれは融合ポリペプチドそれ自体が有用であるのに十分な抗原性を保持する場合であり得る。
【００４５】
抗原性ポリペプチドをスクリーニングし、エピトープ性領域、すなわちポリペプチドの抗原性または免疫原性の原因となるこれらの領域を同定することが可能であることが周知である。そのようなスクリーニングを実施するための方法が、当業界で周知である。こうして、本発明のフラグメントは、１または２以上のそのようなエピトープ性領域を含むべきであり、またはそれらの抗原性／免疫原性特性を保持するそのような領域に十分に類似であるべきである。
【００４６】
こうして、本発明のフラグメントのために、同一性の程度は恐らく重要でなく、なぜなら、これらはここに記載のようなポリペプチド、アナログの特定の部分に１００％同一であり得るからである。
こうして、アナログ、誘導体およびフラグメントとに重要であるのは、これらが、これらが誘導されるタンパク質またはポリペプチドの抗原性／免疫原性の少なくともある程度を保有することである。
【００４７】
またポリペプチドは、それに混合された、ポリペプチド生物学的または薬理学的特性を変化させる他の化合物、すなわち半減期を増加させるためポリエチレングリコール（ＰＥＧ）；精製の容易さのためリーダーまたは分泌アミノ酸配列；ｐｒｅｐｒｏ−またはｐｒｏ−配列；および（多）糖類を含む。
【００４８】
さらに、アミノ酸領域が多型性であると見出される場合のこれらの状況では、ことなるストレプトカッカス菌株の異なるエピトープをより効率的に模倣する１または２以上の特定のアミノ酸を変化することが望ましくあり得る。
【００４９】
さらに、本発明のポリペプチドは、末端、−ＮＨ_２アシル化（例えばアセチル化、またはチオグリコール酸アミド化、末端カルボキシアミド化、例えばアンモニアまたはメチルアミンによる）によって修飾し、支持体または他の分子ヘの連結または結合のための、安定性、増加された疎水性を提供できる。
【００５０】
またポリペプチドフラグメントおよびアナログのへテロおよびホモポリペプチドマルチマーを企図する。これらのポリマー性形態は、例えば架橋剤、例えばアビジン／ビオチン、グルタルアルデヒド（gluteraldehyde）またはジメチルスーパーイミデートで架橋された１または２以上のポリペプチドを含む。そのようなポリマー性形態はまた、組み換えＤＮＡ技法によって生成されたマルチシストロン性ｍＲＮＡから生産された、２または３以上のタンデムまたは逆連続配列を含むポリペプチドを含む。さらなる実施態様では、本発明はまた、本出願の図に定義のような１または２以上のポリペプチドそのフラグメントまたはアナログを含むを含むキメラポリペプチドに関する。
【００５１】
さらなる実施態様では、本発明はまた、配列番号２、またはそのフラグメントまたはアナログから選択される配列を有する、２または３以上のポリペプチドを含むキメラポリペプチドに関する。ただし、該ポリペプチドがキメラポリペプチドを形成するように連結されることを条件とする。
【００５２】
さらなる実施態様では、本発明はまた、配列番号２から選択された配列を有する２または３以上のポリペプチドを含むキメラポリペプチドに関する。ただし、ポリペプチドがキメラポリペプチドを形成するように連結されることを条件とする。
【００５３】
好ましくは、本発明のポリペプチドのフラグメント、アナログまたは誘導体は、少なくとも１つの抗原性領域、すなわち少なくとも１つのエピトープを含む。
【００５４】
抗原性ポリマー（すなわち合成マルチマー）の形成を達成するために、ビスハロアセチル基、ニトロアリールハライドなどを有するポリペプチドを利用し得、ここで該試薬は、チオ基に特異的である。したがって、異なるポリペプチドの２つのメルカプト基の間の連結は、一重結合であり得、または少なくとも２、典型的には少なくとも４、かつ１６を超えないが、通常は約１４を超えないの炭素原子連結基からなり得る。
【００５５】
特定の実施態様では、本発明のポリペプチドフラグメントおよびアナログは、メチオニン（Ｍｅｔ）出発残基を含まない。好ましくは、ポリペプチドは、リーダーまたは分泌配列（シグナル配列）を取りこまない。本発明のポリペプチドのシグナル部分は、確立された分子生物学的技法にしたがって決定し得る。一般に、目的のポリペプチドは、ストレプトコッカス性培養物から単離され、そしてその後に配列決定され、成熟タンパク質の当初残基そしてしたがって成熟ポリペプチドの配列を決定し得る。
【００５６】
ポリペプチドは、それらの開始コドン（メチオニンまたはバリン）なくおよび／または組み換え体ポリペプチドの生産および精製に資するためのこれらのリーダーペプチドなく生産および／または使用することができることが理解される。リーダーペプチドをコードする配列なく遺伝子をクローニングすることは、E. coliの細胞質へポリペプチドを制限し、そしてこれらの回収を容易化することが知られる（Glick, B.R. and Pasternak, J.J.(1998)Manipulation of gene expression in prokaryotes. In''Molecular biotechnology: Principles and applications of recombinant DNA'' 2nd edition, ASM Press, Washington DC, p.109-143）。
【００５７】
本発明の別の側面では、また（ｉ）本発明のポリペプチドを、担体、希釈剤またはアジュバントとともに含む事項の組成物、（ｉｉ）本発明のポリペプチドおよび担体、希釈剤またはアジュバントを含む医薬組成物、（ｉｉｉ）本発明のポリペプチドおよび担体、希釈剤またはアジュバントを含むワクチン、（ｉｖ）宿主において、ストレプトコッカスに対する免疫応答を誘導する方法であって、免疫応答、例えばストレプトコッカスへの保護的免疫応答を誘起するのに免疫原的に有効量の本発明のポリペプチドを投与することによる方法、および（ｖ）ストレプトコッカス感染を予防および／または処置するための方法であって、必要のある宿主に本発明のポリペプチドの予防または治療的量を投与することによる方法、を提供する。
【００５８】
本発明のさらなる側面にしたがってまた、（ｉ）本発明のポリヌクレオチドを、担体、希釈剤またはアジュバントといっしょに含む事項の組成物、（ｉｉ）本発明のポリペプチドおよび担体、希釈剤またはアジュバントを含む医薬組成物、（ｉｉｉ）宿主の、ストレプトコッカスに対する免疫応答を誘導するための方法であって、宿主に、免疫応答、例えばストレプトコッカスへの保護的免疫応答を誘起するための本発明のポリヌクレオチドの免疫原性有効量を投与することによる方法、および特に、（ｉｖ）ストレプトコッカス感染を予防および／または処置するための方法であって、必要のある宿主に本発明のポリペプチドの予防または治療的量を投与することによる方法、を提供する。
【００５９】
免疫化の前に、本発明のポリペプチドをまた、担体タンパク質、例えば破傷風毒素、ジフテリア毒素、Ｂ型肝炎ウイルス表面抗原、小児麻痺ウイルスＶＰ１抗原または任意の他のウイルスまたは細菌性毒素または抗原またはより強い免疫応答の発生を刺激するための任意の好適なタンパク質へカップリングまたはコンジュゲートできる。このカップリングまたはコンジュゲーションを、化学的または遺伝子的に実施できる。ペプチド−担体コンジュゲーションのより詳細な記載は、Van Regenmortel, M.H.V.,Briand J.P., Muller S., Plaue S.. ＜＜Synthetic Plypeptides as antigens＞＞ in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19(ed.) Burdou, R. H. & Van Knippenberg P.H.(1988), Elsevier New Yorkに利用可能である。
【００６０】
さらなる側面にしたがって、１または２以上の本発明のストレプトコッカス性ポリペプチドを、薬学的に許容されるアジュバントとの混合物として含む医薬組成物を提供する。好適なアジュバントは、（１）水中油型エマルジョン製剤、例えばＭＦ５９（登録商標）、ＳＡＦ（登録商標）、Ｒｉｂｉ（登録商標）；（２）フロイントの完全または不完全アジュバント；（３）塩、すなわちＡｌＫ（ＳＯ_４）_２、ＡｌＮａ（ＳＯ_４）_２、ＡｌＮＨ_４（ＳＯ_４）_２、Ａｌ（ＯＨ）_３、ＡｌＰＯ_４、シリカ、カオリン；（４）サポニン誘導体、例えばＳｔｉｍｕｌｏｎ（登録商標）またはそれから生成された粒子例えばＩＳＣＯＭ（免疫刺激複合体）；（５）サイトカイン、例えばインターロイキン、インターフェロン、マクロファージコロニー刺激因子（Ｍ−ＣＳＦ）、腫瘍壊死因子（ＴＮＦ）；（６）他の物質例えばカーボンポリヌクレオチド、すなわちポリＩＣおよびポリＡＵ、解毒化コレラ毒素（ＣＴＢ）および粘膜性免疫の誘導のためのE.coli熱不安定性毒素を含む。アジュバントのより詳細な記載は、M.Z.I Khan et al.によるPharmaceutical Research, vol. 11, No. 1 (1994). pp2-11中のレビューおよびまた Gupta et al., によるVaccine, vol. 13, No. 14, pp1263-1276(1995)の別のレビューおよび引用によりここに全体を含めるＷＯ９９／２４５７８に利用可能である。好ましいアジュバントは、ＱＵＩＬＡ（登録商標）、ＱＳ２１（登録商標）、Ａｌｈｙｄｒｏｇｅｌ（登録商標）およびＡｄｊｕｐｈｏｓ（登録商標）を含む。
【００６１】
本発明の医薬組成物を、注射、急速点滴、鼻咽喉吸収、皮膚吸収によって非経腸にまたは口内または経口投与し得る。
【００６２】
本発明の医薬組成物を、ストレプトコッカス性感染および／またはストレプトコッカス性感染によって仲介される疾患または症状の処置または予防のために使用し、引用によりここに含める、P.R. Murray(Ed, in chef), E.J. Baron, M.A.Phaller, F.C.Tenover and R.H. Yolken. Manual of Clinical Microbiology, ASM Press, Washington, D.C. sixthe edition, 1995, 1482に記載される通りである。１実施態様では、本発明の医薬組成物を、咽頭炎、丹毒および膿痂疹、しょう紅熱、および侵入性疾患、例えば菌血症および壊死性筋膜炎およびまた毒性ショックの予防または処置のために使用する。１実施態様では、本発明の医薬組成物を、ストレプトコッカス感染および／またはストレプトコッカス感染、特にグループＡストレプトコッカス（Streptococcus pyogenes)、グループＢストレプトコッカス（ＧＢＳまたはS. agalactinae)、S. pneumoniae、S. dysgalactinae、S. uberis、S. nocardia並びにStaphylococcus aureusによって仲介される疾患または症状の処置または予防のために使用する。さらなる実施態様では、該ストレプトコッカス感染は、S. pyogenesである。
【００６３】
さらなる実施態様では、本発明はストレプトコッカス感染に感受性の宿主においてストレプトコッカス感染の予防または処置のための方法であって、当該宿主に本発明の組成物の治療または予防的量を投与することを含む方法を提供する。
【００６４】
本出願で使用するように、用語「宿主」は、哺乳動物を含む。さらなる実施態様では、該動物はヒトである。
特定の実施態様では、医薬組成物をストレプト感染のリスクのあるこれらの宿主、例えば幼児、年輩および免疫妥協宿主に投与する。
【００６５】
医薬組成物は好ましくは、約０．００１ないし１００μｇ／ｋｇ（抗原／体重）およびより好ましくは、０．０１ないし１０μｇ／ｋｇおよび最も好ましくは０．１ないし１μｇ／ｋｇの単位用量形態で、免疫化の間約１ないし６週間間隔の間隔で１ないし３回である。
【００６６】
医薬組成物は好ましくは、約０．１μｇないし１０ｍｇ、そしてより好ましくは１ないし１ｍｇそして最も好ましくは１０ないし１００μｇの単位用量形態で、免疫化の間約１ないし６週間間隔の間隔で１ないし３回である。
【００６７】
さらなる側面にしたがって、配列番号２のアミノ酸配列またはそのフラグメントまたはアナログによって特徴付けられるポリペプチドをコードするポリヌクレオチドを提供する。
【００６８】
１実施態様では、ポリヌクレオチドは、本発明のポリペプチドをコードするオープンリーディングフレーム（ＯＲＦ）を含み得る、配列番号１に示されるものである。
【００６９】
図に示されるポリヌクレオチド配列が本発明のポリペプチドをなおもコードする縮重コドンで変化させ得ることが認識される。よって、本発明はさらに、配列間の５０％同一性、１実施態様では配列間の少なくとも７０％同一性、１実施態様では配列間の少なくとも７５％同一性、１実施態様では配列間の少なくとも８０％同一性、１実施態様では配列間の少なくとも８５％同一性、１実施態様では配列間の少なくとも９０％同一性を有する、前記のここのポリヌクレオチド配列（またはその相補的配列）にハイブリダイズするポリヌクレオチドを提供する。さらなる実施態様では、ポリヌクレオチドは、ストリンジェント条件下でハイブリダイズ可能、すなわち少なくとも９５％同一性を有するものである。更なる実施態様では、９７％同一性より高い。
【００７０】
ハイブリダイゼーションのための好適なストリンジェント条件は、当業者によって容易に決定できる（例えば、Sambrook et al., (1989)Molecular cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999) Editea by Ausbel F.M. te al.,john Wiley & Sons, Inc., N.Y.）。
【００７１】
さらなる実施態様では、本発明は、
（ａ）ポリペプチドをコードするＤＮＡ配列または
（ｂ）ポリペプチドをコードするＤＮＡ配列の相補物
にストリンジェント条件下でハイブリダイズするポリヌクレオチドを提供し、ここで当該ポリペプチドは、配列番号２またはそのフラグメントまたはアナログを含む。
【００７２】
さらなる実施態様では、本発明は、
（ａ）ポリペプチドをコードするＤＮＡ配列または
（ｂ）ポリペプチドをコードするＤＮＡ配列の相補物
にストリンジェント条件下でハイブリダイズするポリヌクレオチドを提供し、ここで当該ポリペプチドは配列番号２を含む。
【００７３】
さらなる実施態様では、本発明は、
（ａ）ポリペプチドをコードするＤＮＡ配列または
（ｂ）ポリペプチドをコードするＤＮＡ配列の相補物
にストリンジェント条件でハイブリダイズするポリヌクレオチドを提供し、ここで当該ポリペプチドは、配列番号２またはそのフラグメントまたはアナログを含むポリペプチドからの少なくとも１０連続アミノ酸残基を含む。
【００７４】
さらなる実施態様では、本発明は、
（ａ）ポリペプチドをコードするＤＮＡ配列または
（ｂ）ポリペプチドをコードするＤＮＡ配列の相補物
にストリンジェント条件でハイブリダイズするポリヌクレオチドを提供し、ここで当該ポリペプチドは、配列番号２を含むポリペプチドからの少なくとも１０連続アミノ酸残基を含む。
【００７５】
さらなる実施態様では、ポリヌクレオチドは、配列番号２またはそのフラグメントまたはアナログに示される本発明のポリペプチドをコードするものである。
【００７６】
さらなる実施態様では、ポリヌクレオチドは、本発明のポリペプチドをコードする配列番号１に示されるものまたはそのフラグメントまたはアナログである。
【００７７】
さらなる実施態様では、ポリヌクレオチドは、配列番号２に示される本発明のポリペプチドをコードするものである。
【００７８】
さらなる実施態様では、ポリヌクレオチドは本発明のポリペプチドをコードする配列番号１に示されるものである。
【００７９】
当業者によって容易に認識されるように、ポリヌクレチドはＤＮＡおよびＲＮＡの両方を含む。
【００８０】
本発明はまた、本出願に記載のポリヌクレオチドに相補的なポリヌクレチドを含む。
【００８１】
さらなる側面では、本発明のポリペプチドまたはそのフラグメント、アナログまたは誘導体をコードするポリヌクレオチドを、ＤＮＡ免疫化方法で使用し得る。すなわち、これらを、注射すると複製可能および発現可能であるベクターに取りこませ、それによってインビボで抗原性ポリペプチドを生産し得る。例えばポリヌクレオチドを、真核細胞で機能的であるＣＭＶプロモーターの制御下でプラスミドベクターに取り込ませ得る。好ましくはベクターを筋肉内に注射する。
【００８２】
さらなる側面にしたがって、組み換え技法による本発明のポリペプチドを生産するための方法を提供し、ここで当該ポリペプチドをコードするポリヌクレオチドを宿主細胞へ発現させること、および発現されたポリペプチド産物を回収することによる。代替的にポリペプチドを、確立された合成的化学技法、すなわち完全ポリペプチドを生産するためにライゲートされる、オリゴペプチドの溶液相または固相合成にしたがって生産することができる（ブロックライゲーション）。
【００８３】
ポリヌクレオチドおよびポリペプチドの取得および評価のための一般方法は、以下の引用文献に記載される：引用によりここに含めるSambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al., Johon Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B.A., Humana Press, Totowa, New Jersey, 1997, 490 pages ;Protein Purification, Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan J.E. et al., John Wiley & Sons Inc., New York。
【００８４】
組み換え体生産のために、宿主細胞を、ポリペプチドをコードするベクターでトランスフェクトし、それからプロモーターを活性化する、形質転換体を選択するまたは遺伝子を増幅するために適当に修飾された栄養培地で培養する。好適なベクターは、選択された宿主で生存可能および複製可能であるものであり、そして染色体的、非染色体的および合成ＤＮＡ配列、例えば細菌性プラスミド、ファージＤＮＡ、バキュロウイルス、コウボプラスミド、プラスミドおよびファージＤＮＡの組み合わせに由来するベクターを含む。ポリペプチド配列を、制限酵素を使用して適当な部位えベクターに取りこませ得、その結果それはプロモーター、リボゾーム結合部位（コンセンサス領域またはシャインダルガノ配列）、および所望によりオペレーター（制御エレメント）に作動可能に連結されている。所定の宿主およびベクターに適当である発現制御領域の個々の構成要素を、確立された分子生物学原理にしたがって選択できる（引用によりここに含めるSambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al., Johon Wiley and Sons, Inc. New York）。好適なプロモーターは限定しないが、ＬＴＲまたはＳＶ４０プロモーター、E. coli lac、ｔａｃまたはｔｒｐプロモーターおよびファージラムダＰ_Ｌプロモーターを含む。ベクターは好ましくは複製開始点並びに選択マーカー、すなわちアンプリコン抵抗性遺伝子を取りこむ。好適な細菌性ベクターは、ｐＥＴ、ｐＱＥ７０、ｐＱＥ６０、ｐＱＥ−９、ｐＤ１０ phargescript、ｐｓｉＸ１７４、pbluescript SK、ｐｂｓｋｓ、ｐＮＨ８Ａ、ｐＮＨ１６ａ、ｐＮＨ１８Ａ、ｐＮＨ４６Ａ、ptrc99a、pkk223-3、pkka233-3、ｐＤＲ５４０、ｐＲＩＴ５、および真核ベクターpBlueBacIII、ｐＷＬＮＥＯ、ｐＳＶ２ＣＡＴ、ｐＯＧ４４、ＰＸＴ１、ｐＳＧ、ｐＳＶＫ３、ｐＢＰＶ、ｐＭＳＧおよびｐＳＶＬを含む。宿主細胞は、細菌性、すなわちE. coli、Bacillus subtilis、Streptomyces；真菌性すなわちAspergillus niger、Aspergillus nidulins；コウボすなわちSaccharomycesまたは真核性すなわちＣＨＯ、ＣＯＳであり得る。
【００８５】
培養でポリペプチドを発現すると、細胞を典型的には、遠心分離によって収集し、次いで物理学的または化学的手段によって分解し（もし発現されたポリペプチドが、培地中に分泌されていないならば）、そして得られた粗抽出物が保持され、所望のポリペプチドを単離する。粗培地または細胞溶解物からのポリペプチドの精製を、ポリペプチドの特性に依存して確立された技法によって達成し得、すなわち硫酸アンモニウムまたはエタノール沈降、酸抽出、アニオンまたはカチオン荷電クロマトグラフィー、ホスホセルロースクロマトグラフィー、疎水性相互作用クロマトグラフィー、ヒドロキシルアパタイトクロマトグラフィーおよびレクチンクロマトグラフィーを使用する。最終精製を、ＨＰＬＣを使用して達成し得る。
【００８６】
ポリペプチドを、リーダーまたは分泌配列を有してまたは有しないで発現させ得る。前者の場合、リーダーを、翻訳後プロセシングを使用して除去し（引用によりここに含めるUS4431739;US4425437およびUS4338397参照）または発現されたポリペプチドを精製するに続いて化学的に除去し得る。
【００８７】
さらなる側面にしたがって、本発明のストレプトコッカス性ポリペプチドを、ストレプトコッカス感染、特にS. pyogenes感染についての診断試験において使用し得る。いくつかの診断方法、例えば生物学的サンプル中のストレプトコッカス生物を検出することが可能であり、以下の手法に従う：
ａ）宿主から生物学的サンプルを取得する
ｂ）抗体または本発明のストレプトコッカスポリペプチドと反応性のそのフラグメントと、生物学的サンプルをインキュベートし混合物を形成する、および
ｃ）混合物中の特異的に結合した抗体または結合フラグメントを検出し、それはストレプトコッカスの存在を指摘する。
【００８８】
代替的に、当該抗体を含むまたは含むと疑われる生物学的サンプル中のストレプトコッカス抗原に特異的な抗体の検出方法は、以下のように実施され得る：
ａ）宿主から生物学的サンプルを取得する、
ｂ）１または２以上の本発明のストレプトコッカスポリペプチドまたはそのフラグメントを、生物学的サンプルとインキュベートし、混合物を形成する、および
ｃ）混合物中の特異的に結合した抗原または結合フラグメントを検出し、それはストレプトコッカスの存在を指摘する。
当業者は、この診断試験が、免疫的試験、例えばエライザアッセイ（ＥＬＩＳＡ）、放射性免疫アッセイまたはラテックス接合アッセイを含む幾つかの形態をとり、タンパク質に特異的な抗体が生物中に存在するか本質的に決定し得る。
【００８９】
本発明のポリペプチドをコードするＤＮＡ配列をまた使用し、そのような細菌を含むまたと疑われる生物学的サンプル中のストレプトコッカスの存在を検出することにおける使用のためのＤＮＡプローブを設計し得る。この発明の検出方法は
ａ）宿主から生物学的サンプルを取得する、
ｂ）本発明のポリペプチドまたはそのフラグメントをコードするＤＮＡ配列を有する１または２以上のＤＮＡプローブを、生物学的サンプルとインキュベートし、混合物を形成する、および
ｃ）混合物中の特異的に結合したＤＮＡプローブを検出し、それはストレプトコッカスの存在を指摘する、
を含む。
【００９０】
本発明のＤＮＡプローブはまた、例えばストレプトコッカス感染を診断する方法として、ポリメラーゼ連鎖反応を使用して、サンプル中の循環するストレプトコッカス、すなわちS. pyogenes核酸を検出するために使用し得る。プローブを、慣行技法を使用して合成し得、そして固相上に固定化し、または検出可能標識で標識し得る。この適用のための好ましいＤＮＡプローブは、本発明のS. pyogenesポリペプチドの少なくとも約６連続ヌクレオチドに相補的な配列を有するオリゴマーである。
【００９１】
宿主中のストレプトコッカスの検出のためのさらなる診断方法は、
ａ）本発明のポリペプチドまたはそのフラグメントと反応性の抗体を、検出可能標識で標識すること
ｂ）該標識した抗体または標識されたフラグメントを、宿主に投与すること、および
ｃ）宿主中の特異的に結合した標識された抗体または標識されたフラグメントを検出し、それはストレプトコッカスの存在を指摘する、
を含む。
【００９２】
さらなる側面にしたがって、本発明は、ストレプトコッカス性感染の処置および／または予防のための抗体の使用（用途）を提供する。
【００９３】
本発明のさらなる側面は、診断のため、および特にストレプトコッカス感染の処置における特異的抗体の生産のための免疫原として本発明のストレプトコッカスポリペプチドの使用である。好適な抗体は、適当なスクリーニング方法を使用して、例えば特定の抗体の、試験モデルでストレプトコッカス感染に対して受動的に保護する能力を測定することによって決定し得る。動物モデルの１例は、ここでの例で記載するマウスモデルである。抗体は全体抗体または抗原に結合するそのフラグメントであり得、そして任意の免疫グロブリンクラスに属し得る。該抗体またはフラグメントは、動物起源の、具体的には哺乳動物起源の、より具体的にはマウス、ラットまたはヒト起源のものであり得る。それは天然抗体またはそのフラグメント、または望まれれば、組換え抗体または抗体フラグメントであり得る。用語組換え抗体または抗体フラグメントは、分子生物学的技法を使用して生産された抗体または抗体フラグメントを意味する。抗体または抗体フラグメントは、ポリクローナルまたは好ましくはモノクローナル抗体であり得る。それは、S. pyogenesポリペプチドに付随するエピトープのある数に特異的であり得るが、好ましくは１つに特異的である。
【００９４】
本発明のさらなる側面は、受動的免疫化のための本発明のポリペプチドに指向化された（directed）抗体の使用である。本出願で記載された抗体を使用し得る。
【００９５】
本発明のさらなる側面は、免疫化の方法であって、ここに本発明のポリペプチドによって惹起された(raised)抗体を、受動的免疫化を提供するのに十分量で宿主に投与する。
【００９６】
さらなる実施態様では、本発明は、ストレプトコッカス性感染の予防または治療的処置のための医薬の製造における医薬組成物の使用を提供する。
【００９７】
更なる実施態様では、本発明は、ストレプトコッカス感染の検出または診断のための本発明のポリペプチドを含むキットを提供する。
【００９８】
特に定義しない場合、ここの全ての技術および科学用語は、本発明の属する当業者によって普通に理解されるのと同じ意味を有する。すべての刊行物、特許出願、特許および他のここに述べた引用は、引用により全体を含める。抵触の場合は、定義を含む本明細書が制御する。加えて材料、方法および例は、例示のみであり、限定を意図しない。
【００９９】
実施例１
この例は、ＢＶＨ−Ｐ７遺伝子および対応するポリペプチドのクローニングおよび分子特徴を示す。
【０１００】
S.pyogenes BVH-P7（配列番号１）遺伝子のコード領域を、血清型Ｍ１ S. pyogenes菌株ＡＴＣＣ／００２９４のゲノムＤＮＡからＰＣＲによって増幅する（Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, CA）。表１に示す、制限部位ＮｄｅＩ（ＣＡＴＡＴＧ）およびＮｏｔＩ（ＧＣＧＧＣＣＧＣ）の付加のための塩基伸長物を含ませる以下のオリゴヌクレオチドプライマーを使用する：ＤＭＡＲ２９３およびＤＭＡＲ２９４。ＰＣＲ産物を、製造者の指示（Chatsworth, CA）にしたがって、QIAgenからのQIAquickゲル抽出キットを使用してアガロースゲルから精製し、そしてＮｄｅＩおよびＮｏｔＩで消化する（Amersham Pharmacia Biotech Inc, Baie d'Urfe, Canada）。ｐＥＴ−２１（＋）ベクター（Novagen, Madison, WI)を、ＮｄｅＩおよびＮｏｔＩで消化し、そしてQIAgen(Chatsworth, CA)からのQIAquickゲル抽出キットを使用してアガロースゲルから精製した。ＮｄｅＩ−ＮｏｔＩ ＰＣＲ産物を、ＮｄｅＩ−ＮｏｔＩ ｐＥＴ−２１ｂ（＋）発現ベクターにライゲートさせた。ライゲートされた産物を、Simanisの方法（Hanahan, D. DNA Cloning, 1085, D.M. Glover(ed), pp.109-135）にしたがって
【化１】

に移入した。ＢＶＨ−Ｐ７遺伝子を含む組み換え体ｐＥＴ−２１ｂ（＋）プラスミド（ｒｐＥＴ２１ｂ（＋））をQIAgenプラスミドキット（Chatsworth, CA）を使用して精製し、そしてＤＮＡインサートを配列決定した（Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, CA）。
【０１０１】
表１ ＰＣＲ増幅またはS. pyogenes BVE-P7遺伝子のために使用されるオリゴヌクレオチドプライマー
【表２】

【０１０２】
ＢＶＨ−Ｐ７のオープンリーディングフレーム（ＯＲＦ）停止コドン（ＴＡＡ）を含む３０２７ｂｐは、６．１８の予測されたｐＩおよび１１１４９４．４４Ｄａの予測された分子量を有する１００８アミノ酸残基ポリペプチドをコードする。PSORTIIソフトウェア（Real World Computing Partnership(http://psort.nibb.ac.jp)を使用する予測されたアミノ酸残基配列（配列番号２）の分析は、アスパラギンとグルタミン残基の間に位置する切断部位で終わる２１アミノ酸残基シグナルペプチド
【化２】

の存在を示唆した。アミノ酸残基配列の分析は、残基９７４６と９８１の間に位置する細胞壁アンカーモチーフ（ＬＰＸＴＧＸ）の存在をあきらかにした。
【０１０３】
ＢＶＨ−Ｐ７（配列番号１）遺伝子のＰＣＲ増幅によって存在を確認するために、以下の４つの血清学的に明瞭なS. pyogenes菌株を使用した：血清型Ｍ１ S. pyogenes菌株ＡＴＣＣ７００２９４および血清型M3 S.pyogenes菌株ＡＴＣＣ１２３８４を、アメリカンタイプカルチャーコレクション(Rockville, MD)から取得した；血清型Ｍ６ S. pyogenes SPY67臨床的単離株は、Centre de recherche en infectiologie du Centre hospitalier de l' universite Laval, Sainte-Foyによって提供された;およびマウスから当初単離されたS. pyogenes菌株Ｂ５１４は、University of Alabama, BirminghamからSusan Hollingsheadによって提供された。E. coli菌株ＸＬ１−Ｂｌｕｅ ＭＲＦを、ネガティブコントロールとしてこれらの実験で使用した。染色体性ＤＮＡを、以前記載されたようにそれぞれのS. pyogenes菌株から単離した（Jayarao EM et al., 1991. J. Clin. Microbiol. 29:2774-2778）。ＢＶＨ−Ｐ７（配列番号１）遺伝子を、４つのS. pyogenes菌株から精製されたゲノムＤＮＡからＰＣＲ（Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, Ca）によって、そしてコントロールE. coli菌株をオリゴヌクレオチドプライマーＤＭＡＲ２９３およびＤＭＡＲ２９４（表１）を使用して増幅した。ＰＣＲを、４５秒間の９５℃、４５秒間の５０℃および２分間の７２℃の３０サイクルおよび７分間の７２℃の最終伸長期間で実施した。ＰＣＲ産物を、１％アガロースゲル中でサイズ分画化し、そして臭化エチジウム染色によって可視化した。これらのＰＣＲ増幅の結果を表２に表す。増幅産物の分析は、ＢＶＨ−Ｐ７（配列番号１）遺伝子が試験された４つのS. pyogenes菌株のすべてのゲノム中に存在した。そのような産物を、コントロールE. coli ＤＮＡがこれらのオリゴヌクレオチドプライマーと同一のＰＣＲ増幅に服するとき検出した。
【０１０４】
表２ ４つの血清学的に明瞭なS. pyogenes菌株のゲノムにおける、ＰＣＲ増幅によるS. pyogenes BVH-P7遺伝子の同定
【表３】

【０１０５】
実施例２
この例は、ＣＭＶプラスミドｐＣＭＶ−ＧＨにおけるS. pyogenes BVH-P7遺伝子のクローニングを示す。
S.pyogenesタンパク質のＤＮＡコード領域を、プラスミドベクターｐＣＭＶ−ＧＨ（Tang et al., Nature, 1992, 356:152）のサイトメガロウイルス（ＣＭＶ）プロモーターの転写制御下にあるヒト成長ホルモン（ｈＧＨ）遺伝子の下流の相(phase）に挿入した。該ＣＭＶプロモーターは、E. coli細胞において非機能性プラスミドであるが、真核細胞においてプラスミドを投与すると活性である。該ベクターはまた、アンシピリン抵抗性遺伝子を取りこんだ。
【０１０６】
リーダーペプチド領域を欠くＢＶＨ−Ｐ７（配列番号１）遺伝子のコード領域を、表１に記載の、制限部位ＢａｍＨＩ（ＧＧＡＴＣＣ）およびＳａｌＩ（ＧＴＣＧＡＣ）の付加のための塩基伸長物を含んだオリゴヌクレオチドプライマーＤＭＡＲ４８０ａおよびＤＭＡＲ４８１ａを使用して、血清型Ｍ１ S. pyogenes菌株ＡＴＣＣ７００２９４のゲノムＤＮＡからＰＣＲ（Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, CA)によって増幅した。ＰＣＲ産物を、QIAgen(chatsworth, CA)からのQIAquickゲル抽出キットを使用してアガロースゲルから精製し、制限酵素（Amersham Pharmacia Biotech Inc, Baied'Uefe, Canada）で消化した。ｐＣＭＶ−ＧＨベクター（Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas)を、ＢａｍＨＩおよびＳａｌＩで消化し、そしてＱＩＡｇｅｎ（Chatsworth, CA)からのQIAquickゲル抽出キットを使用してアガロースゲルから精製した。ＢａｍＨＩ−ＳａｌＩ ＤＮＡフラグメントを、ＢａｍＨＩ−ＳａｌＩ−ｐＣＭＶ−ＧＨベクターにライゲートし、ＣＭＶプロモーターの制御下のｈＧＨ−ＢＶＨ−Ｐ７融合タンパク質を創出した。ライゲートした産物を、Simanisの方法（Hanahan, D. DNA Cloning, 1985, D.M. Glover(ed), pp. 109-135）にしたがって、
【化３】

に形質転換した。組み換え体ｐＣＭＶプラスミドを、ＱＩＡｇｅｎプラスミドキット（Chatsworth, CA）を使用して精製しそしてＤＮＡインサートのヌクレオチド配列を、ＤＮＡ配列決定によって確認した。
【０１０７】
実施例３
この例は、S. pyogenes BVH-P7タンパク質抗原への免疫応答を誘起するＤＮＡの使用を記載する。
８匹のメスＢＡＬＢ／ｃマウス（Charles River, St-Constant, Quebec, canada）の群を、５０μｇの顆粒球−マクロファージコロニー刺激因子（ＧＭ−ＣＳＦ）を発現するプラスミドｐＣＭＶ−ＧＨ−ＧＭ−ＣＳＦ(Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas)の存在下の、ＢＶＨ−Ｐ７（配列番号１）遺伝子をコードする組み換え体ｐＣＭＶ−ＧＨの５０μｇでの２または３週間間隔での、１００μｌ３回の筋肉内注射によって免疫化した。コントロールとして、マウスの群に、５０μｇのｐＣＭＶ−ＧＨ−ＧＭ−ＣＳＦの存在下の５０μｇのｐＣＭＶ−ＧＨを注射した。血液サンプルを、それぞれの免疫化に先立ちおよび第３注射の７日後に眼窩洞（orbital sinus）から収集し、コート抗原としてBVH-P7 Hisタグ付加標識したS. pyogenes組み換え体タンパク質を使用してＥＬＩＳＡによって決定した。このBVH-P7 Hisタグ付加標識したS. pyogenes組み換え体タンパク質の生産および精製を実施例４に提示する。
【０１０８】
実施例４
この例は、S. pyogenes ＢＶＨ−Ｐ７組み換え体タンパク質の生産および精製を示す。
ＢＶＨ−Ｐ７（配列番号１）遺伝子での組換え体ｐＥＴ−２１ｂ（＋）プラスミドを使用し、エレクトロポレーション（Gene Pulser II apparratus, BIO-RAD Labs, Mississauga, Canada）によって、E. coli菌株Ｔｕｎｅｒ（ＤＥ３）(F^-ompT hsdS_B(r^- _Bm^- _B)gel dcm lacYI(DE))(Novagen, Madison, WI)を形質転換した。E. coliのこの菌株において、組み換え体タンパク質の発現を制御するＴ７プロモーターは、遺伝子が、イソプロピル−β−ｄ−チオ−ガラクトピラノシド（ＩＰＴＧ）によって誘導可能であるｌａｃプロモーターの制御下である、Ｔ７ ＲＮＡポリメラーゼ（λＤＥ３プロファージ上に存在する）によって特異的に認識される。形質転換体Ｔｕｎｅｒ（ＤＥ３）／ｒｐＥＴ２１（＋）を、ｍｌあたり１００μｇのカルベニシリン（Sigma-Aldrich Canada Ltd., Oakville, Canada）を含むＬＢ培地（ペプトン１０ｇ／Ｌ、コウボエキス５ｇ／Ｌ、ＮａＣｌ１０ｇ／Ｌ）中で２５０ｒｐｍで撹拌しつつ３７℃で、Ａ_５００が０．６の値に達するまで成長させた。ＢＶＨ−Ｐ７ Ｈｉｓタグ付加S. pyogenes組み換え体タンパク質の生産を誘導するために、細胞を０．１ｍＭの最終濃度のＩＰＴＧの存在下もう３時間インキュベートした。５００ｍｌ培養物から誘導された細胞を、遠心分離によってペレット化し、そして−７０℃で凍結した。
【０１０９】
不溶性分画またはＩＰＴＧで誘導したＴｕｎｅｒ（ＤＥ３）／ｒｐＥＴ２１ｂ（＋）からのＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質の精製を、Ｈｉｓ・Ｔａｇ配列（６連続ヒスチジン残基）の、Ｈｉｓ・Ｂｉｎｄ金属キレート化樹脂上に固定化された２価カチオンに結合する特性に基づいてアフィニティークロマトグラフィーによって実施した。簡単には、ＩＰＴＧで誘導された５００ｍＬ培養物から取得されたペレット化細胞を、６Ｍ グアニジン−ＨＣｌを含む溶解バッファー（２０ｍＭ Ｔｒｉｓ、５００ｍＭ ＮａＣｌ、１０ｍＭ イミダゾール、ｐＨ７．９）中に再懸濁し、超音波処理し、そして１２０００Ｘｇで２０分間遠心分離し、細胞残骸を除去した。上清を、Ｎｉ−ＮＴＡアガロース樹脂（Qiagen, Mississauga, Ontario, Canada）と45分間4℃でインキュベートした。BVH-P7Ｈｉｓタグ付加S. pyogenes組み換え体タンパク質を、６Ｍ グアニジノ−ＨＣｌおよび２５０ｍＭ イミダゾール−５００ｍＭ ＮａＣｌ−２０ｍＭ Ｔｒｉｓ、ｐＨ７．９を含む溶液で樹脂から溶出させた。サンプルからの塩およびイミダゾールの除去を、１０ｍＭ Ｔｒｉｓおよび０．９％ ＮａＣｌ、ｐＨ７．９で終夜４℃に対する透析によってなした。組み換え体タンパク質の量を、ＭｉｃｒｏＢＣＡ（Pierce, Rockford, Illinois）によって評価した。
【０１１０】
実施例５
この例は、ヒト血清および、S. pyogenes抗原性調製品での免疫化後のマウスから収集した血清でのＢＶＨ−Ｐ７ Ｈｉｓタグ付加S. pyogenes組み換え体タンパク質の反応性を示す。
表３に示すように、精製されたＨｉｓタグ付加ＢＶＨ−Ｐ７組換え体タンパク質を、通常の血清の貯留物中に存在する抗体によるイムノブロットにおいて認識した。これは重要な結果であり、なぜならそれはS. pyogenesと通常接触するヒトが、そのタンパク質へ特異的である抗体を発生させることを明かに示すからである。これらの特定のヒト抗体は、S. pyogenes感染に対する保護を暗示し得る。加えて、イムノブロットはまた、マウスを致死チャレンジに対して保護する、膜タンパク質富化されたS. pyogenes抗原性調製品で、免疫化されたマウスから収集された血清が、ＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質を認識する抗体をまた発生させたことを明かにした。この結果は、このタンパク質が感染に対してマウスを保護したS. pyogenes 抗原性調製品中に存在すること、およびこのストレプトコッカス性タンパク質が、対応するＨｉｓタグ付加組換え体タンパク質と反応する抗体を誘導したことを指摘する。
【０１１１】
表３ ヒト血清および、ＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質でS. pyogenes抗原性調製品で免疫化後の、マウスから収集された血清のイムノブロットの反応性
【表４】

^１実施例７に記載されたように生産および精製されたＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質を使用し、イムノブロットを実施した。
^２ＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質の分子量を、ＳＤＳ−ＰＡＧＥ後に評価した。
^３健康ヒトボランティアから収集された血清を一緒に貯留し、そして１／５００に希釈しイムノブロットを実施した。
^４膜タンパク質富化S. pyogenes抗原性調製品で免疫化後収集されたマウス血清を貯留し、そして１／５００希釈し、イムノブロットを実施した。これらのマウスは、致死S. pyogenesチャレンジに対して保護された。
【０１１２】
実施例６
この例は、インタクトのストレプトコッカス性細胞の表面のS. pyogenes ＢＶＨ−Ｐ７タンパク質の抗体への接近可能性を示す。
細菌を、０．５％コウボエキス(Difco Laboratories)および０．５％ペプトンエキス（Merck, Darmstadt, Germany）を有するTood Hewitt(TH)培地（Difco Laboratories, Detroit, MI）中で、３７℃で８％ＣＯ_２雰囲気中で成長させ、０．６００のＯＤ_４９０ｎｍを与えた（〜１０^８ＣＦＵ／ｍｌ）。抗ＢＶＨ−Ｐ７またはコントロール血清の希釈品を次いで添加し、そして細胞に結合させ、これを２時間４℃でインキュベートした。サンプルをブロッキングバッファー（２％ウシ血清アルブミン（ＢＳＡ）を含む燐酸バッファー生食水（ＰＢＳ））で４回洗浄し、それからブロッキングバッファー中に希釈した１ｍｌのヤギフルオレセイン（ＦＩＴＣ）コンジュゲート抗マウスＩｇＧ＋ＩｇＭを添加した。室温での追加的６０分のインキュベーション後、サンプルをブロッキングバッファーで４回洗浄し、そしてＰＢＳバッファー中の０．２５％ホルムアルデヒドで１８−２４時間４℃で固定化した。細胞をＰＢＳバッファーで２回洗浄し、そしてＰＢＳバッファーの５００μｌ中に再懸濁した。細胞を、フローサイトメトリー（Epics(登録商標)XL; Beckman Coulter, Inc.)によって分析するまで暗黒で４℃で保持した。サンプルあたり１万のインタクトなS. pyogenes細胞を分析し、そして結果を標識された細胞の存在および蛍光指数として表現した。蛍光指数を、コントロールマウス血清について取得された蛍光値によって割った、免疫血清でストレプトコッカス性細胞を標識した後得られたメジアン蛍光値として算出した。１の蛍光値は、インタクトのストレプトコッカス性細胞の表面の抗体の結合がないことを指摘した。
【０１１３】
ＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質で免疫化された８匹のマウスから収集した血清を、サイトフルオロメトリーによって分析し、そして結果を表４に表す。精製されたＢＶＨ−Ｐ７ Ｈｉｓタグ付加タンパク質で免疫化されたマウスから収集された血清の全ては、試験した異種性（ＡＴＣＣ１２３８４；血清型Ｍ３）S. pyogenes菌株上の、それらの対応する表面曝露エピトープを効率的に認識するＢＶＨ−Ｐ７に特異的抗体を含んだ。蛍光指数は１０から１８まで変化した。分析した１００００このS. pyogenes細胞のうち９７％より多くが、ＢＶＨ−Ｐ７特異的抗血清中に存在する抗体で標識された。これらの血清をまた貯留し、そして以下のS. pyogenes菌株と反応させた：血清型Ｍ１ S. pyogenes菌株ＡＴＣＣ７００２９４、血清型Ｍ３および血清型Ｍ１８ S. pyogenes 菌株ＡＴＣＣ１２３５７を、アメリカンタイプカルチャーコレクション（Rockville, MD, USA）から取得した；血清型Ｍ６ S. pyogenes ＳＰＹ６９およびＭ２ S. pyogenes ＳＰＹ６８臨床的単離株は、Centre de recherche en infectiologie du Centre hospitalier de 1'universite Laval, Sainte-Foyによって提供された。精製されたＨｉｓタグ付加組換え体ＢＶＨ−Ｐ７タンパク質で免疫化後収集された血清の貯留物中に存在するＢＶＨ−Ｐ７−特異的抗体は、４から９までの間の蛍光指数を有するこれらのストレプトコッカス性菌株のそれぞれの細菌の表面に付着した。反対に、非免疫化または偽免疫化（sham-immunized）マウスから収集した貯留物または血清を使用したとき、ストレプトコッカス性細胞の標識は認められなかった。これらの観察は明白に、ＢＶＨ−Ｐ７タンパク質が表面に接近可能であり、ここにそれは抗体によって容易に認識されることができることを実証する。抗S. pyogenes抗体は、S. pyogenes感染に対する保護において重要な役割を演ずることを示した。
【０１１４】
表４ S. pyogenes ＡＴＣＣ１２３８４菌株（血清型Ｍ３）のインタクトの細胞の表面のＢＶＨ−Ｐ７特異的抗体の付着の評価
【表５】

^１マウスＳ１ないしＳ８を皮下に、QuilAアジュバント（Cedarlane Laboratories, Hornby, Canada)の１０μｇと混合した２０μｇの精製されたＢＶＨ−Ｐ７組換え体タンパク質の２０μｇで３週間間隔で３回注射した。血清を１／５０希釈した。
^２蛍光指数を、コントロールマウスについて取得された蛍光値によって分割された免疫血清でストレプトコッカス性細胞を標識した後取得されたメジアン蛍光値として算出した。１の蛍光値は、インタクトなストレプトコッカス性細胞の表面の抗体の結合がないことを示した。
^３分析した１００００細胞からのストレプトコッカス標識した細胞の％
^４非免疫化または偽免疫化されたマウスから収集された血清を、貯留１／５０希釈し、そしてこのアッセイのためのネガティブコントロールとして使用した。
２０μｇの精製されたストレプトコッカス性組換えＭタンパク質、すなわち周知の表面タンパク質で免疫化されたマウスから取得された血清を、１／２００希釈し、そしてアッセイのためのポジティブコントロールとして使用した。
【０１１５】
実施例７
この例は、ウサギハイパー免疫血清でのマウスの受動的免疫化によって誘導された胎児S. pyogenes 感染に対する保護を示す。
ニュージーランドウサギ（Charles River Laboratories, St-Constant, Canada）を皮下に、実施例４に記載のように生産および精製し、そしてAlhydrogelアジュバント（Superfos Biosector a/s）に吸着させたたＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質の、５０μｇおよび１００μｇで多重部位に注射した。ウサギをＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質で３週間間隔で３回免疫化した。血液サンプルを、第３注射の３週間後に収集した。該血清中に存在する抗体を、４０％飽和硫酸アンモニウムを使用する沈降によって精製した。１０匹のメスのＣＤ−１マウス（Charles River）の群を静脈内に、ＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質で免疫化されたウサギ、または関連のないコントロール組み換え体タンパク質で免疫化されたウサギから収集された精製された血清の５００μｌで注射した。１８時間後、マウスを近似的に２Ｘ１０^７ ＣＦＵのタイプ３S. pyogenes菌株ＡＴＣＣ１２３８４でチャレンジした。S. pyogens チャレンジ接種物のサンプルを、アガープレート上に置き、ＣＦＵを決定し、そしてチャレンジ用量を確認した。死亡を、５日の期間について記録した。
【０１１６】
実施例８
この例は、精製された組み換え体ＢＶＨ−Ｐ７タンパク質での免疫化によって誘導される胎児S. pyogenes感染に対するマウスの保護を示す。
８匹のメスのＢａｌｂ／ｃ（Charles River, St-Constant, Quebec, Canada）の群を皮下に、１０μｇのＱｕｉｌＡアジュバント（Cedarlane Laboratories Ltd, Hornby, Canada）の存在下のアフィニティ精製されたＢＶＨ−Ｐ７ Ｈｉｓタグ付加組換え体タンパク質の２０μｇ、またはコントロールとしてＰＢＳ中のＱｕｉｌＡアジュバント単独で２週間間隔で３回免疫化した。血液サンプルを、それぞれの免疫化の前の第１、第１４および２８日に、第３注射の２週間後（第４２日）に、眼窩洞（orbital sinus）から収集した。１週間後、マウスを、近似的に２ｘ１０６ ＣＦＵのタイプ３S. pyogenes菌株ＡＴＣＣ１２３８４でチャレンジした。S. pyogenes チャレンジ接種物のサンプルを、血液アガープレート上に置き、ＣＦＵを決定しそしてチャレンジ用量を確認した。精製組換え体ＢＶＨ−Ｐ７タンパク質で免疫化された、８匹のマウスのうち４匹は、致死チャレンジに対して保護され、アジュバント単独を受容したマウスのわずか１２％（１／８）と対比される（表１）。
【０１１７】
表５ 組換え体ＢＶＨ−Ｐ７タンパク質の、ＧＡＳ菌株ＡＴＣＣ １２３８４（タイプ３）に対する保護を誘起する能力
【表６】

[0001]
Field of the invention
The present invention relates to a polypeptide of Streptococcus pyogenes (Group A Streptococcus), which can be used to prevent, diagnose and / or treat streptococcal infection.
[0002]
Background of the Invention
Streptococci is a Gram-positive bacterium that is found on the cell surface and is differentiated by group-specific carbohydrate antigens A through O. S. pyogenes isolates are further distinguished by type-specific M protein antigens. It is an important infectious agent that can vary highly in both molecular weight and sequence. In fact, more than 80 M protein types were identified based on antigenic differences.
[0003]
S. pyogenes is the causative agent of many widespread infectious forms, including pharyngitis, erysipelas and impetigo, scarlet fever and invasive diseases such as bacteremia and necrotizing fasciitis. Recent invasive disease relapses have been reported in many countries, including North American and European countries. Although the organism is susceptible to antibiotics, high attack rates and rapid onset of spoilage result in high incidence and mortality.
[0004]
Efforts have focused on infectious agents, such as type-specific M proteins, to develop vaccines that protect the host from S. pyogenes infection. However, it was found that the amino-terminal portion of the M protein induced cross-reactive antibodies that reacted with human myocardium, tropomyosin, myosin, and vimentin, which could be involved in autoimmune diseases. Others used recombinant techniques to produce complex hybrid proteins containing the amino-terminal peptide of the M protein from various serotypes. However, safe vaccines containing all S. pyogenes serotypes are highly complex to produce and standardize.
[0005]
In addition to serotype-specific antigens, other S. pyogenes proteins have generated interest as vaccine candidates. C5a peptidase, which is expressed by at least the S. pyogenes 40 serotype, has been shown to be immunogenic in mice, but has a limited ability to reduce the level of nasopharyngeal metastatic proliferation. there were. Other investigators have also focused on streptococcal spirogenic exotoxins, which appear to play an important role in the pathogenesis of the infection. Immunization with these proteins prevented the lethal symptoms of toxic shock, but did not prevent colonization.
[0006]
The University of Oklahoma has set up a genome sequencing project for S. pyogenes strain M1 GAS (http: //dnal/chem.ou.edu/strep.html).
[0007]
Thus, there remains an unmet need for S. pyogens antigens, which may be vaccine components used for prevention and / or treatment of S. pyogenes infection.
[0008]
Summary of the Invention
According to one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0009]
According to one aspect, the invention relates to a polypeptide comprising the amino acid sequence SEQ ID NO: 2 or a fragment or analog thereof.
[0010]
In other aspects, the polypeptides encoded by the polynucleotides of the invention, pharmaceutical compositions, vectors comprising the polynucleotides of the invention operably linked to expression control regions, and host cells transfected with the vectors And methods for producing a polypeptide comprising culturing the host cell under conditions suitable for expression.
[Brief description of the drawings]
[0011]
FIG. 1 shows the DNA sequence of the BVH-P7 gene from serotype M1S. Pyoegenes strain ATCC 700294; SEQ ID NO: 1. The underlined part of the sequence indicates the region encoding the leader peptide.
FIG. 2 shows the amino acid sequence BVH-P7 protein from serotype M1S. Pyogenes strain ATCC 700294; SEQ ID NO: 2. The underlined sequence indicates a 21 amino acid residue leader peptide.
FIG. 3-1 shows MacVector sequence analysis software (version 6) of the deduced amino acid sequence of the BVH-P7 open reading frame derived from Spy74, Spy70, Spy69, Spy68, Spy60, ATCC12357, ATCC700294S.pyogenes strain. 5 shows a comparison by using the program Clustal W from 5). Below the alignment, there is a consensus line where the * and. Letters indicate the same and similar amino acid residues, respectively.
FIG. 23 shows MacVector sequence analysis software (version 6.5) of the deduced amino acid sequence of the BVH-P7 open reading frame derived from Spy74, Spy70, Spy69, Spy68, Spy60, ATCC12357, ATCC700294S.pyogenes strain. 3) shows a comparison by using the program Clustal W from). Below the alignment, there is a consensus line where the * and. Letters indicate the same and similar amino acid residues, respectively.
FIG. 3-3 shows MacVector sequence analysis software (version 6) of the deduced amino acid sequence of the BVH-P7 open reading frame derived from Spy74, Spy70, Spy69, Spy68, Spy60, ATCC12357, ATCC700294S.pyogenes strain. 5 shows a comparison by using the program Clustal W from 5). Below the alignment, there is a consensus line where the * and. Letters indicate the same and similar amino acid residues, respectively.
FIG. 3-4 shows MacVector sequence analysis software (version 6.x) of the deduced amino acid sequence of BVH-P7 open reading frame derived from Spy74, Spy70, Spy69, Spy68, Spy60, ATCC12357, ATCC700294S.pyogenes strain. 5 shows a comparison by using the program Clustal W from 5). Below the alignment, there is a consensus line where the * and. Letters indicate the same and similar amino acid residues, respectively.
[0012]
Detailed description of the invention
The present invention provides purified and isolated polynucleotides encoding streptococcal polypeptides that can be used to diagnose, prevent, and / or treat streptococcal infection.
[0013]
According to one aspect, the present invention provides an isolated polypeptide that encodes a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0014]
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0015]
According to one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0016]
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0017]
According to one aspect, the invention relates to a polypeptide characterized by an amino acid sequence comprising SEQ ID NO: 2 or a fragment or analog thereof.
According to one aspect, the invention provides a polynucleotide encoding a portion having an epitope of a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0018]
According to one aspect, the invention relates to an epitope-bearing portion of a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0019]
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID NO: 2.
[0020]
According to one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID NO: 2.
[0021]
According to one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID NO: 2.
[0022]
According to one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID NO: 2.
[0023]
According to one aspect, the invention relates to a polypeptide characterized by the amino acid sequence comprising SEQ ID NO: 2.
[0024]
According to one aspect, the present invention provides a polynucleotide encoding an epitope-bearing portion of a polypeptide comprising SEQ ID NO: 2.
[0025]
According to one aspect, the invention relates to an epitope-bearing portion of a polypeptide comprising SEQ ID NO: 2.
[0026]
According to one aspect, the present invention provides:
(A) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(B) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(C) a polynucleotide encoding a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(D) a polynucleotide encoding a polypeptide capable of producing an antibody having binding specificity for a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof
(E) a polynucleotide encoding a portion having a epitope of a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(F) a polynucleotide comprising a sequence selected from SEQ ID NO: 1 or a fragment or analog thereof
(G) a polynucleotide that is complementary to the polynucleotide of (a), (b), (c), (d), (e) or (f)
An isolated polynucleotide comprising a polypeptide selected from the group consisting of:
[0027]
According to one aspect, the present invention provides:
(A) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2
(B) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2
(C) a polynucleotide encoding a polypeptide comprising the sequence selected from SEQ ID NO: 2
(D) nucleotides encoding a polypeptide capable of raising an antibody having binding specificity for a polypeptide comprising a sequence selected from SEQ ID NO: 2
(E) a polynucleotide encoding a portion having an epitope of a polypeptide comprising a sequence selected from SEQ ID NO: 2
(F) a polynucleotide comprising a sequence selected from SEQ ID NO: 1
(G) a polynucleotide that is complementary to the polynucleotide of (a), (b), (c), (d), (e) or (f)
An isolated polynucleotide comprising a polynucleotide selected from the group consisting of:
[0028]
According to one aspect, the present invention provides:
(A) a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof
(B) a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof;
(C) a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof
(D) a polypeptide capable of raising an antibody having binding specificity to a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof
(E) a portion having an epitope of a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof
(F) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the N-terminal methionine residue is deleted;
(G) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the secreted amino acid sequence is deleted;
An isolated polypeptide comprising a polypeptide selected from:
[0029]
According to one aspect, the present invention provides:
(A) a polypeptide having at least 70% identity to the second polypeptide comprising SEQ ID NO: 2,
(B) a polypeptide having at least 95% identity to the second polypeptide comprising SEQ ID NO: 2
(C) Polypeptide containing sequence number 2
(D) a polypeptide capable of raising an antibody having binding specificity to the polypeptide comprising SEQ ID NO: 2
(E) a portion having an epitope of a polypeptide including SEQ ID NO: 2
(F) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the N-terminal methionine is deleted;
(G) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the secreted amino acid sequence is deleted;
An isolated polypeptide comprising a polypeptide selected from:
[0030]
One skilled in the art will recognize that the present invention encodes DNA molecules, ie, analogs, such as mutants, variants, homologs, and derivatives of such polypeptides, as described herein in this patent application. , Polynucleotides and their complementary sequences. The present invention also includes RNA molecules corresponding to the DNA molecules of the present invention. In addition to DNA and RNA molecules, the invention includes the corresponding polypeptides and monospecific antibodies that specifically bind to such polypeptides.
[0031]
In a further embodiment, the polypeptide according to the invention is antigenic.
In a further embodiment, the polypeptide according to the invention is immunogenic.
In a further embodiment, a polypeptide according to the invention is capable of eliciting immune responsiveness in a host.
[0032]
In a further embodiment, the invention also relates to a polypeptide capable of raising an antibody having binding specificity for a polypeptide of the invention as defined above.
[0033]
An antibody that “has binding specificity” is an antibody that recognizes and binds a selected polypeptide, but does not substantially recognize and bind other molecules in a sample, eg, a biological sample. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.
[0034]
According to the present invention, "protection" in a biological study is defined by a significant increase in survival curve, rate or duration. Statistical analysis using the Log rank test to compare survival curves, respectively, and using Fisher's exact method to compare survival and days to death, calculate P values and compare between the two groups May be useful to determine if the differences are significant in a statistical determination. A P value of 0.05 is considered insignificant.
[0035]
In an additional aspect of the invention, there is provided an antigenic / immunogenic fragment of a polypeptide of the invention, or an analog thereof.
[0036]
Fragments of the present invention should contain one or more such epitope regions, or should be sufficiently similar to such regions to retain their antigenic / immunogenic properties. Thus, for fragments according to the present invention, the degree of identity is probably irrelevant, as they can be 100% identical to a particular portion of the polypeptide or an analog thereof, as described herein. The present invention further provides fragments having at least 10 contiguous amino acid residues from the polypeptide sequences of the present invention. In one embodiment, at least 15 contiguous amino acid residues. In one embodiment, at least 20 contiguous amino acid residues.
[0037]
The major problem is again that the fragments retain antigenic / immunogenic properties.
Those skilled in the art will recognize that analogs of the polypeptides of the present invention will also find use in the context of the present invention, ie, as antigenic / immunogenic materials. Thus, for example, proteins or polypeptides containing one or more additions, deletions, substitutions, or the like are included in the invention.
[0038]
As used herein, a "fragment", "analog" or "derivative" of a polypeptide of the invention is one in which one or more amino acid residues can be natural or non-natural (conservative) or non-conservative. These polypeptides include those that have been substituted with amino acid residues (preferably conserved). In one embodiment, the derivatives and analogs of the peptides of the invention have about 70% identity to these sequences or fragments thereof shown in the figures. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 80% identity. In a further embodiment, polypeptides will have greater than 85% identity. In a further embodiment, polypeptides will have greater than 90% identity. In a further embodiment, polypeptides will have greater than 95% identity. In a further embodiment, polypeptides will have greater than 99% identity. In a further embodiment, an analog of a polypeptide of the invention has less than about 20 amino acid residue substitutions, modifications or deletions, and more preferably less than 10.
[0039]
These substitutions have the least effect on the secondary structure and hydropathic properties of the polypeptide. Preferred substitutions are those known in the art as conservative, that is, the substituted residues have both physical or chemical properties, such as hydrophobicity, size, charge, or functionality. These include substitutions such as those described by Dayhoff, M in Atlas of Protein Sequence and Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785, 1989. For example, natural or unnatural amino acids belonging to any of the following groups represent conservative changes:
[Table 1]

Preferred substitutions also include substitution of the D-enantiomer for the corresponding L-amino acid.
[0040]
In an alternative approach, an analog of the polypeptide of the invention comprises the substitutions disclosed in FIG.
In an alternative approach, the analog may be a fusion protein that contains a moiety that makes purification easier, for example, by efficiently tagging the desired polypeptide. It may be necessary to remove the "tag", or it may be when the fusion polypeptide retains itself sufficient antigenicity to be useful.
[0041]
The percentage of homology is defined as the percentage of similarity or conservation of amino acid types in addition to the percentage of identity.
[0042]
In one embodiment, an analog or polypeptide of the invention has about 70% identity to these sequences or fragments thereof shown in the figures. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 75% homology. In a further embodiment, polypeptides will have greater than 80% homology. In a further embodiment, polypeptides will have greater than 85% homology. In a further embodiment, polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, an analog of a polypeptide of the invention has less than about 20 amino acid residue substitutions, modifications or deletions, and more preferably less than 10.
[0043]
Amino acid sequences can be compared using a program, such as the CLUSTAL program. The program compares amino acid sequences and finds an optimal alignment by inserting spaces in any sequence as appropriate. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for optimal alignment. Programs such as BLASTx align the longest stretch of similar sequence and assign a value to the fit. Thus, it is possible to obtain a comparison in which several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated by the present invention.
[0044]
In an alternative approach, the analog or derivative can be a fusion polypeptide that incorporates a moiety that makes purification easier, e.g., by efficiently tagging the desired protein or polypeptide, removing the "tag" Or it may be where the fusion polypeptide itself retains sufficient antigenicity to be useful.
[0045]
It is well known that antigenic polypeptides can be screened to identify epitopic regions, ie, those regions responsible for the antigenicity or immunogenicity of the polypeptide. Methods for performing such screening are well known in the art. Thus, a fragment of the invention should include one or more such epitopic regions, or should be sufficiently similar to such regions to retain their antigenic / immunogenic properties. is there.
[0046]
Thus, for the fragments of the present invention, the degree of identity is probably not important, as they can be 100% identical to a particular portion of the polypeptide, analog as described herein.
Thus, what is important for analogs, derivatives and fragments is that they retain at least some of the antigenic / immunogenic properties of the protein or polypeptide from which they are derived.
[0047]
The polypeptide may also be mixed with other compounds that alter the biological or pharmacological properties of the polypeptide, ie, polyethylene glycol (PEG) to increase half-life; leader or secreted amino acids for ease of purification. Sequences; prepro- or pro-sequences; and (poly) saccharides.
[0048]
Further, in these situations where the amino acid region is found to be polymorphic, it may be desirable to change one or more specific amino acids that more efficiently mimic different epitopes of different Streptococcus strains. possible.
[0049]
Further, the polypeptides of the present invention may comprise a terminal, -NH₂Modification by acylation (eg, acetylation, or thioglycolic acid amidation, terminal carboxyamidation, eg, with ammonia or methylamine) to increase stability, for coupling or attachment to a support or other molecule. Can provide high hydrophobicity.
[0050]
Also contemplated are hetero and hetero polypeptide multimers of polypeptide fragments and analogs. These polymeric forms include, for example, one or more polypeptides cross-linked with a cross-linking agent, such as avidin / biotin, gluteraldehyde or dimethyl superimidate. Such polymeric forms also include polypeptides containing two or more tandem or reverse contiguous sequences produced from multicistronic mRNAs produced by recombinant DNA techniques. In a further embodiment, the present invention also relates to chimeric polypeptides comprising one or more polypeptides as defined in the figures of this application, including fragments or analogs thereof.
[0051]
In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides having a sequence selected from SEQ ID NO: 2, or a fragment or analog thereof. Provided that the polypeptides are linked so as to form a chimeric polypeptide.
[0052]
In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides having a sequence selected from SEQ ID NO: 2. Provided that the polypeptides are linked so as to form a chimeric polypeptide.
[0053]
Preferably, a fragment, analog or derivative of a polypeptide of the invention comprises at least one antigenic region, ie at least one epitope.
[0054]
To achieve the formation of an antigenic polymer (ie, a synthetic multimer), a polypeptide having a bishaloacetyl group, a nitroaryl halide, etc. may be utilized, wherein the reagent is specific for a thio group. Thus, the link between the two mercapto groups of different polypeptides may be a single bond, or at least 2, typically at least 4, and usually no more than about 16 carbon atoms. It can consist of a linking group.
[0055]
In certain embodiments, polypeptide fragments and analogs of the present invention do not include a methionine (Met) starting residue. Preferably, the polypeptide does not incorporate a leader or secretory sequence (signal sequence). The signal portion of a polypeptide of the present invention can be determined according to established molecular biology techniques. Generally, the polypeptide of interest can be isolated from a streptococcal culture and subsequently sequenced to determine the initial residues of the mature protein and thus the sequence of the mature polypeptide.
[0056]
It is understood that polypeptides can be produced and / or used without their initiation codon (methionine or valine) and / or without these leader peptides to contribute to the production and purification of recombinant polypeptides. Cloning a gene without the sequence encoding the leader peptide is known to limit the polypeptide to the cytoplasm of E. coli and facilitate their recovery (Glick, BR and Pasternak, JJ (1998) Manipulation of gene expression in prokaryotes. In "Molecular biotechnology: Principles and applications of recombinant DNA" 2nd edition, ASM Press, Washington DC, p.109-143).
[0057]
In another aspect of the invention, also a composition comprising (i) a polypeptide of the invention together with a carrier, diluent or adjuvant, (ii) a medicament comprising a polypeptide of the invention and a carrier, diluent or adjuvant. A composition, (iii) a vaccine comprising the polypeptide of the invention and a carrier, diluent or adjuvant, (iv) a method for inducing an immune response against Streptococcus in a host, comprising the step of protecting the immune response against Streptococcus, eg, Streptococcus. A method by administering an immunogenically effective amount of a polypeptide of the invention to elicit a response, and (v) a method for preventing and / or treating a Streptococcus infection, comprising administering to a host in need thereof By administering a prophylactic or therapeutic amount of a polypeptide of the invention.
[0058]
According to a further aspect of the invention also, (i) a composition comprising a polynucleotide of the invention together with a carrier, diluent or adjuvant, (ii) a polypeptide of the invention and a carrier, diluent or adjuvant. A pharmaceutical composition comprising: (iii) a method for inducing an immune response of a host against Streptococcus, the method comprising the step of eliciting an immune response in the host, eg, a protective immune response to Streptococcus; A method by administering an immunogenically effective amount, and in particular, (iv) a method for preventing and / or treating a Streptococcus infection, wherein the host in need is treated with a prophylactic or therapeutic amount of a polypeptide of the present invention. Or a method of administering
[0059]
Prior to immunization, the polypeptides of the invention may also comprise a carrier protein, such as tetanus toxin, diphtheria toxin, hepatitis B virus surface antigen, pediatric palsy virus VP1 antigen or any other virus or bacterial toxin or antigen or more. It can be coupled or conjugated to any suitable protein to stimulate the development of a strong immune response. This coupling or conjugation can be performed chemically or genetically. A more detailed description of peptide-carrier conjugation can be found in Van Regenmortel, MHV, Briand JP, Muller S., Plaue S .. << Synthetic Plypeptides as antigens >> in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (ed. .) Available in Burdou, RH & Van Knippenberg PH (1988), Elsevier New York.
[0060]
According to a further aspect, there is provided a pharmaceutical composition comprising one or more streptococcal polypeptides of the invention as a mixture with a pharmaceutically acceptable adjuvant. Suitable adjuvants are (1) oil-in-water emulsion formulations, such as MF59®, SAF®, Ribi®; (2) complete or incomplete adjuvants of Freund's; AlK (SO₄)₂, AlNa (SO₄)₂, AlNH₄(SO₄)₂, Al (OH)₃, AlPO₄(4) saponin derivatives such as Stimulon (R) or particles produced therefrom such as ISCOM (immunostimulating complex); (5) cytokines such as interleukin, interferon, macrophage colony stimulating factor (M- CSF), tumor necrosis factor (TNF); (6) other substances such as carbon polynucleotides, ie poly IC and poly AU, detoxified cholera toxin (CTB) and E. coli heat instability for induction of mucosal immunity Contains toxins. A more detailed description of adjuvants can be found in Pharmaceutical Research, vol. 11, No. 1 (1994) by MZI Khan et al., And also in Vaccine, vol. 13, No. 1, by Gupta et al., Pp2-11. 14, pp 1263-1276 (1995), which is available in WO 99/24578, which is hereby incorporated by reference in its entirety. Preferred adjuvants include QUILA®, QS21®, Alhydrogel®, and Adjuphos®.
[0061]
The pharmaceutical compositions of the present invention may be administered parenterally or buccally or orally by injection, rapid infusion, nasopharyngeal absorption, dermal absorption.
[0062]
The pharmaceutical compositions of the present invention are used for the treatment or prevention of streptococcal infections and / or diseases or conditions mediated by streptococcal infections and are incorporated herein by reference. PR Murray (Ed, in chef), EJ Baron, MAPhaller, FCTenover and RH Yolken. Manual of Clinical Microbiology, ASM Press, Washington, DC sixth edition, 1995, 1482. In one embodiment, the pharmaceutical composition of the present invention is used for the prevention or treatment of pharyngitis, erysipelas and impetigo, rash, and invasive diseases such as bacteremia and necrotizing fasciitis and also toxic shock. Used for In one embodiment, the pharmaceutical composition of the present invention is used for treating Streptococcus infection and / or Streptococcus infection, especially Group A Streptococcus pyogenes, Group B Streptococcus (GBS or S. agalactinae), S. pneumoniae, S. dysgalactinae, S. uberis, S. nocardia and Staphylococcus aureus for the treatment or prevention of diseases or conditions. In a further embodiment, the Streptococcus infection is S. pyogenes.
[0063]
In a further embodiment, the present invention is a method for preventing or treating Streptococcus infection in a host susceptible to Streptococcus infection, comprising administering to the host a therapeutic or prophylactic amount of a composition of the present invention. I will provide a.
[0064]
As used in this application, the term "host" includes mammals. In a further embodiment, the animal is a human.
In certain embodiments, the pharmaceutical composition is administered to those hosts at risk for streptococcal infection, such as infants, seniors, and immune compromised hosts.
[0065]
The pharmaceutical composition is preferably administered in a unit dosage form of about 0.001 to 100 μg / kg (antigen / body weight) and more preferably 0.01 to 10 μg / kg and most preferably 0.1 to 1 μg / kg. One to three times at intervals of about 1 to 6 weeks during the formation.
[0066]
The pharmaceutical composition is preferably in a unit dosage form of about 0.1 μg to 10 mg, and more preferably 1 to 1 mg and most preferably 10 to 100 μg, for 1 to 3 weeks at intervals of about 1 to 6 weeks during immunization. Times.
[0067]
According to a further aspect, there is provided a polynucleotide encoding a polypeptide characterized by the amino acid sequence of SEQ ID NO: 2, or a fragment or analog thereof.
[0068]
In one embodiment, the polynucleotide is as set forth in SEQ ID NO: 1, which can include an open reading frame (ORF) encoding a polypeptide of the invention.
[0069]
It will be appreciated that the polynucleotide sequences shown in the figures may be altered at degenerate codons which still encode the polypeptides of the present invention. Thus, the invention further provides 50% identity between sequences, in one embodiment at least 70% identity between sequences, in one embodiment at least 75% identity between sequences, and in one embodiment at least 80% identity between sequences. Hybridizes to the herein described polynucleotide sequence (or its complementary sequence), having at least 85% identity between sequences, in one embodiment at least 85% identity between sequences, in one embodiment. A polynucleotide is provided. In a further embodiment, the polynucleotides are hybridizable under stringent conditions, ie, have at least 95% identity. In a further embodiment, greater than 97% identity.
[0070]
Suitable stringent conditions for hybridization can be readily determined by one skilled in the art (eg, Sambrook et al., (1989) Molecular cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor, NY; Current Protocols in Molecular). Biology, (1999) Editea by Ausbel FM te al., John Wiley & Sons, Inc., NY).
[0071]
In a further embodiment, the invention provides a method comprising:
(A) a DNA sequence encoding a polypeptide or
(B) the complement of the DNA sequence encoding the polypeptide
Provides a polynucleotide that hybridizes under stringent conditions, wherein the polypeptide comprises SEQ ID NO: 2 or a fragment or analog thereof.
[0072]
In a further embodiment, the invention provides a method comprising:
(A) a DNA sequence encoding a polypeptide or
(B) the complement of the DNA sequence encoding the polypeptide
Provides a polynucleotide that hybridizes under stringent conditions, wherein the polypeptide comprises SEQ ID NO: 2.
[0073]
In a further embodiment, the invention provides a method comprising:
(A) a DNA sequence encoding a polypeptide or
(B) the complement of the DNA sequence encoding the polypeptide
Provides a polynucleotide that hybridizes under stringent conditions, wherein the polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof.
[0074]
In a further embodiment, the invention provides a method comprising:
(A) a DNA sequence encoding a polypeptide or
(B) the complement of the DNA sequence encoding the polypeptide
Provides a polynucleotide that hybridizes under stringent conditions, wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising SEQ ID NO: 2.
[0075]
In a further embodiment, the polynucleotide encodes a polypeptide of the invention set forth in SEQ ID NO: 2 or a fragment or analog thereof.
[0076]
In a further embodiment, the polynucleotide is that shown in SEQ ID NO: 1 or a fragment or analog thereof encoding a polypeptide of the invention.
[0077]
In a further embodiment, the polynucleotide encodes a polypeptide of the invention set forth in SEQ ID NO: 2.
[0078]
In a further embodiment, the polynucleotide is as set forth in SEQ ID NO: 1, which encodes a polypeptide of the invention.
[0079]
As will be readily appreciated by those skilled in the art, polynucleotides include both DNA and RNA.
[0080]
The invention also includes polynucleotides complementary to the polynucleotides described in the present application.
[0081]
In a further aspect, a polynucleotide encoding a polypeptide of the present invention or a fragment, analog or derivative thereof can be used in a DNA immunization method. That is, they can be incorporated into a vector that is replicable and expressible upon injection, thereby producing an antigenic polypeptide in vivo. For example, a polynucleotide can be incorporated into a plasmid vector under the control of a CMV promoter that is functional in eukaryotic cells. Preferably, the vector is injected intramuscularly.
[0082]
According to a further aspect, there is provided a method for producing a polypeptide of the invention by recombinant techniques, wherein a polynucleotide encoding the polypeptide is expressed in a host cell and the expressed polypeptide product is recovered. By doing. Alternatively, polypeptides can be produced according to established synthetic chemistry techniques, ie, solution or solid phase synthesis of oligopeptides that are ligated to produce complete polypeptides (block ligation).
[0083]
General methods for obtaining and evaluating polynucleotides and polypeptides are described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed, Cold Spring Harbor, NY, incorporated herein by reference. 1989; Current Protocols in Molecular Biology, Edited by Ausubel FM et al., Johon Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White BA, Humana Press, Totowa, New Jersey, 1997, 490 pages; Protein Purification, Principles and Practices, Scopes RK, Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan JE et al., John Wiley & Sons Inc., New York.
[0084]
For recombinant production, host cells are transfected with a vector encoding the polypeptide and then in a nutrient medium suitably modified to activate the promoter, select transformants or amplify the gene. Incubate. Suitable vectors are those that are viable and replicable in the chosen host, and that contain chromosomal, non-chromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, plasmids and phage. Includes vectors derived from combinations of DNA. The polypeptide sequence can be incorporated into an appropriate site vector using restriction enzymes so that it is activated by a promoter, a ribosome binding site (consensus region or Shine-Dalgarno sequence), and optionally an operator (control element). It is connected as possible. Individual components of the expression control regions that are appropriate for a given host and vector can be selected according to established molecular biology principles (Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed, incorporated herein by reference). Cold Spring Harbor, NY, 1989; Current Protocols in Molecular Biology, Edited by Ausubel FM et al., Johon Wiley and Sons, Inc. New York). Suitable promoters include, but are not limited to, LTR or SV40 promoter, E. coli lac, tac or trp promoter and phage lambda P_LIncludes promoter. The vector preferably incorporates an origin of replication as well as a selectable marker, the amplicon resistance gene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pkk223-3, pkka233-3, pDR540, pRIT5. Includes eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, PXT1, pSG, pSVK3, pBPV, pMSG and pSVL. The host cell can be bacterial, ie, E. coli, Bacillus subtilis, Streptomyces; fungal, ie, Aspergillus niger, Aspergillus nidulins; yeast, Saccharomyces or eukaryotic, ie, CHO, COS.
[0085]
Upon expression of the polypeptide in culture, the cells are typically collected by centrifugation and then degraded by physical or chemical means (if the expressed polypeptide is not secreted into the medium, ) And the resulting crude extract retained, isolating the desired polypeptide. Purification of the polypeptide from the crude medium or cell lysate can be achieved by established techniques depending on the properties of the polypeptide, i.e., ammonium sulfate or ethanol precipitation, acid extraction, anion or cation charge chromatography, phosphocellulose chromatography. Use chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification can be achieved using HPLC.
[0086]
The polypeptide may be expressed with or without a leader or secretory sequence. In the former case, the leader may be removed using post-translational processing (see US4431739; US4425437 and US4338397, incorporated herein by reference) or chemically removed following purification of the expressed polypeptide.
[0087]
According to a further aspect, a Streptococcal polypeptide of the invention may be used in a diagnostic test for Streptococcal infection, especially S. pyogenes infection. Several diagnostic methods are possible, for example the detection of Streptococcus organisms in biological samples, according to the following procedure:
a) obtaining a biological sample from the host
b) incubating a biological sample with an antibody or a fragment thereof reactive with a Streptococcus polypeptide of the invention to form a mixture; and
c) detecting specifically bound antibody or binding fragment in the mixture, which indicates the presence of Streptococcus.
[0088]
Alternatively, a method for detecting an antibody specific for a Streptococcus antigen in a biological sample containing or suspected of containing the antibody may be performed as follows:
a) obtaining a biological sample from the host;
b) incubating one or more Streptococcus polypeptides of the invention or a fragment thereof with a biological sample to form a mixture, and
c) detecting specifically bound antigen or binding fragment in the mixture, which indicates the presence of Streptococcus.
One of skill in the art will appreciate that this diagnostic test may take several forms, including immunological tests, such as an ELISA, a radioimmunoassay or a latex conjugation assay, to determine whether antibodies specific for the protein are present in the organism. Can be determined.
[0089]
DNA sequences encoding the polypeptides of the present invention can also be used to design DNA probes for use in detecting the presence of Streptococcus in biological samples containing or suspected of containing such bacteria. The detection method of the present invention
a) obtaining a biological sample from the host;
b) incubating one or more DNA probes having a DNA sequence encoding a polypeptide of the invention or a fragment thereof with a biological sample to form a mixture, and
c) detecting the specifically bound DNA probe in the mixture, which indicates the presence of Streptococcus;
including.
[0090]
The DNA probes of the present invention can also be used to detect circulating Streptococcus, ie, S. pyogenes nucleic acids in a sample using the polymerase chain reaction, for example, as a method of diagnosing Streptococcus infection. Probes may be synthesized using conventional techniques, and may be immobilized on a solid phase or labeled with a detectable label. Preferred DNA probes for this application are oligomers having a sequence complementary to at least about 6 consecutive nucleotides of a S. pyogenes polypeptide of the present invention.
[0091]
Additional diagnostic methods for the detection of Streptococcus in a host include:
a) Labeling an antibody reactive with the polypeptide of the present invention or a fragment thereof with a detectable label.
b) administering the labeled antibody or labeled fragment to a host;
c) detecting specifically bound labeled antibody or labeled fragment in the host, which indicates the presence of Streptococcus;
including.
[0092]
According to a further aspect, the present invention provides the use (use) of an antibody for the treatment and / or prevention of streptococcal infection.
[0093]
A further aspect of the invention is the use of a Streptococcus polypeptide of the invention as an immunogen for diagnosis, and in particular for the production of specific antibodies in the treatment of Streptococcus infection. Suitable antibodies can be determined using appropriate screening methods, for example, by measuring the ability of a particular antibody to passively protect against Streptococcus infection in a test model. One example of an animal model is the mouse model described in the examples herein. Antibodies can be whole antibodies or fragments thereof that bind to an antigen, and can belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin, more particularly of mouse, rat or human origin. It can be a natural antibody or fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment refers to an antibody or antibody fragment produced using molecular biological techniques. The antibody or antibody fragment can be a polyclonal or, preferably, a monoclonal antibody. It may be specific for a certain number of epitopes associated with the S. pyogenes polypeptide, but is preferably specific for one.
[0094]
A further aspect of the invention is the use of antibodies directed to the polypeptide of the invention for passive immunization. The antibodies described in the present application may be used.
[0095]
A further aspect of the invention is a method of immunization, wherein an antibody raised by a polypeptide of the invention is administered to a host in an amount sufficient to provide passive immunization.
[0096]
In a further embodiment, the present invention provides the use of a pharmaceutical composition in the manufacture of a medicament for the prevention or therapeutic treatment of a Streptococcal infection.
[0097]
In a further embodiment, the present invention provides a kit comprising a polypeptide of the present invention for detecting or diagnosing a Streptococcus infection.
[0098]
Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0099]
Example 1
This example demonstrates the cloning and molecular characterization of the BVH-P7 gene and corresponding polypeptide.
[0100]
The coding region of the S. pyogenes BVH-P7 (SEQ ID NO: 1) gene is amplified by PCR from genomic DNA of serotype M1 S. pyogenes strain ATCC / 00294 (Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, CA). The following oligonucleotide primers containing the base extensions for addition of the restriction sites NdeI (CATATG) and NotI (GCGGCCGC), shown in Table 1, are used: DMAR293 and DMAR294. The PCR product is purified from agarose gel using the QIAquick gel extraction kit from QIAgen according to the manufacturer's instructions (Chatsworth, CA) and digested with NdeI and NotI (Amersham Pharmacia Biotech Inc, Baie d'Urfe). , Canada). The pET-21 (+) vector (Novagen, Madison, WI) was digested with NdeI and NotI and purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA). The NdeI-NotI PCR product was ligated into an NdeI-NotI pET-21b (+) expression vector. The ligated product was prepared according to the method of Simanis (Hanahan, D. DNA Cloning, 1085, D.M. Glover (ed), pp.109-135).
Embedded image

Was imported. Recombinant pET-21b (+) plasmid containing the BVH-P7 gene (rpET21b (+)) was purified using the QIAgen plasmid kit (Chatsworth, CA) and the DNA insert was sequenced (Taq Dye Deoxy Terminator Cycle). Sequencing kit, ABI, Foster City, CA).
[0101]
Table 1 Oligonucleotide primers used for PCR amplification or S. pyogenes BVE-P7 gene
[Table 2]

[0102]
3027 bp, including the open reading frame (ORF) stop codon (TAA) of BVH-P7, encodes a 1008 amino acid residue polypeptide having a predicted pi of 6.18 and a predicted molecular weight of 111494.44 Da. Analysis of the predicted amino acid residue sequence (SEQ ID NO: 2) using PSORTII software (Real World Computing Partnership (http://psort.nibb.ac.jp) revealed that the cleavage located between the asparagine and glutamine residues 21 amino acid residue signal peptide ending at the site
Embedded image

Suggested the existence of. Analysis of the amino acid residue sequence revealed the presence of a cell wall anchor motif (LPXTGX) located between residues 9746 and 981.
[0103]
The following four serologically distinct S. pyogenes strains were used to confirm their presence by PCR amplification of the BVH-P7 (SEQ ID NO: 1) gene: serotype M1 S. pyogenes strain ATCC 700294 and serotype M3. S. pyogenes strain ATCC 12384 was obtained from the American Type Culture Collection (Rockville, MD); the serotype M6 S. pyogenes SPY67 clinical isolate was from the Center de recherche en infectiologie du Center hospitalier de l'universite Laval, Sainte- S. pyogenes strain B514, originally isolated from mice, was provided by Susan Hollingshead from the University of Alabama, Birmingham. E. coli strain XL1-Blue MRF was used in these experiments as a negative control. Chromosomal DNA was isolated from each S. pyogenes strain as previously described (Jayarao EM et al., 1991. J. Clin. Microbiol. 29: 2774-2778). The BVH-P7 (SEQ ID NO: 1) gene was purified from genomic DNA purified from four S. pyogenes strains by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, Ca) and control E. coli strains were oligonucleotide primers. Amplification was performed using DMAR293 and DMAR294 (Table 1). PCR was performed with 30 cycles of 95 ° C for 45 seconds, 50 ° C for 45 seconds and 72 ° C for 2 minutes and a final extension period of 72 ° C for 7 minutes. PCR products were size fractionated in a 1% agarose gel and visualized by ethidium bromide staining. Table 2 shows the results of these PCR amplifications. Analysis of the amplification products showed that the BVH-P7 (SEQ ID NO: 1) gene was present in the genome of all four S. pyogenes strains tested. Such products were detected when control E. coli DNA was subjected to the same PCR amplification as these oligonucleotide primers.
[0104]
Table 2 Identification of the S. pyogenes BVH-P7 gene by PCR amplification in the genomes of four serologically distinct S. pyogenes strains
[Table 3]

[0105]
Example 2
This example shows the cloning of the S. pyogenes BVH-P7 gene in the CMV plasmid pCMV-GH.
The human growth hormone (hGH) gene under the transcriptional control of the cytomegalovirus (CMV) promoter of the plasmid vector pCMV-GH (Tang et al., Nature, 1992, 356: 152), using the DNA coding region of the S. pyogenes protein Into the downstream phase. The CMV promoter is a non-functional plasmid in E. coli cells, but is active upon administration of the plasmid in eukaryotic cells. The vector also incorporated the ancipiline resistance gene.
[0106]
Oligonucleotide primers containing a coding region of the BVH-P7 (SEQ ID NO: 1) gene lacking the leader peptide region and a base extension for addition of restriction sites BamHI (GGATCC) and SalI (GTCGAC) as shown in Table 1. It was amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, CA) from genomic DNA of serotype M1 S. pyogenes strain ATCC 700294 using DMAR480a and DMAR481a. PCR products were purified from agarose gels using the QIAquick gel extraction kit from QIAgen (chatsworth, CA) and digested with restriction enzymes (Amersham Pharmacia Biotech Inc, Baied'Uefe, Canada). The pCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas) was digested with BamHI and SalI and a QIAquick gel extraction kit from QIAgen (Chatsworth, CA) was used. And purified from agarose gel. The BamHI-SalI DNA fragment was ligated into the BamHI-SalI-pCMV-GH vector to create an hGH-BVH-P7 fusion protein under the control of a CMV promoter. The ligated product was prepared according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover (ed), pp. 109-135).
Embedded image

Was transformed. Recombinant pCMV plasmid was purified using the QIAgen plasmid kit (Chatsworth, CA) and the nucleotide sequence of the DNA insert was confirmed by DNA sequencing.
[0107]
Example 3
This example describes the use of DNA to elicit an immune response to the S. pyogenes BVH-P7 protein antigen.
Groups of eight female BALB / c mice (Charles River, St-Constant, Quebec, canada) were treated with the plasmid pCMV-GH-GM-CSF expressing 50 μg of granulocyte-macrophage colony stimulating factor (GM-CSF) ( 2 or 50 μg of recombinant pCMV-GH encoding the BVH-P7 (SEQ ID NO: 1) gene in the presence of the Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas). Immunization was performed by three intramuscular injections of 100 μl at three week intervals. As a control, a group of mice was injected with 50 μg of pCMV-GH in the presence of 50 μg of pCMV-GH-GM-CSF. Blood samples were collected from the orbital sinus prior to each immunization and 7 days after the third injection and ELISA using BVH-P7 His-tagged S. pyogenes recombinant protein as coat antigen. Determined by The production and purification of this BVH-P7 His-tagged recombinant S. pyogenes protein is presented in Example 4.
[0108]
Example 4
This example illustrates the production and purification of a recombinant S. pyogenes BVH-P7 protein.
The E. coli strain Tuner was prepared by electroporation (Gene Pulser II apparratus, BIO-RAD Labs, Mississauga, Canada) using the recombinant pET-21b (+) plasmid with the BVH-P7 (SEQ ID NO: 1) gene. (DE3) (F^-ompT hsdS_B(r^- _Bm^- _B) gel dcm lacYI (DE)) (Novagen, Madison, WI). In this strain of E. coli, the T7 promoter controlling the expression of the recombinant protein is under the control of the lac promoter, whose gene is inducible by isopropyl-β-d-thio-galactopyranoside (IPTG). , T7 RNA polymerase (present on λDE3 prophage). The transformant Tuner (DE3) / rpET21 (+) was added to LB medium (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) containing 100 μg of carbenicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml. At 37 ° C. while stirring at 250 rpm in₅₀₀Grew to a value of 0.6. Cells were incubated for another 3 hours in the presence of 0.1 mM final concentration of IPTG to induce production of BVH-P7 His-tagged S. pyogenes recombinant protein. Cells derived from the 500 ml culture were pelleted by centrifugation and frozen at -70 ° C.
[0109]
Purification of BVH-P7 His-tagged recombinant protein from Tuner (DE3) / rpET21b (+) induced by insoluble fraction or IPTG was performed by His-Bind metal of His.Tag sequence (6 consecutive histidine residues). Performed by affinity chromatography based on the property of binding to divalent cations immobilized on the chelating resin. Briefly, pelleted cells obtained from a 500 mL culture induced with IPTG were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9) containing 6 M guanidine-HCl, Sonicate and centrifuge at 12000 × g for 20 minutes to remove cell debris. The supernatant was incubated with Ni-NTA agarose resin (Qiagen, Mississauga, Ontario, Canada) for 45 minutes at 4 ° C. The BVH-P7His-tagged S. pyogenes recombinant protein was eluted from the resin with a solution containing 6 M guanidino-HCl and 250 mM imidazole-500 mM NaCl-20 mM Tris, pH 7.9. Removal of salts and imidazole from the samples was done by dialysis against 4 mM overnight at 10 mM Tris and 0.9% NaCl, pH 7.9. The amount of recombinant protein was assessed by MicroBCA (Pierce, Rockford, Illinois).
[0110]
Example 5
This example shows the reactivity of BVH-P7 His-tagged recombinant S. pyogenes protein in human serum and serum collected from mice after immunization with an S. pyogenes antigenic preparation.
As shown in Table 3, purified His-tagged BVH-P7 recombinant protein was recognized in an immunoblot with antibodies present in a pool of normal serum. This is an important result because it is clear that humans normally in contact with S. pyogenes develop antibodies that are specific for the protein. These particular human antibodies may imply protection against S. pyogenes infection. In addition, the immunoblot also shows that sera collected from immunized mice with a membrane protein-enriched S. pyogenes antigenic preparation protects mice against lethal challenge with BVH-P7 His-tag. It was revealed that antibodies that recognize the additional recombinant protein were also generated. This result indicates that this protein is present in the S. pyogenes antigenic preparation that protected mice against infection, and that this streptococcal protein induced antibodies that reacted with the corresponding His-tagged recombinant protein. Point out that
[0111]
Table 3 Immunoblot reactivity of sera collected from mice after immunization with human serum and S. pyogenes antigenic preparation with BVH-P7 His-tagged recombinant protein
[Table 4]

¹Immunoblot was performed using BVH-P7 His-tagged recombinant protein produced and purified as described in Example 7.
²The molecular weight of the BVH-P7 His-tagged recombinant protein was evaluated after SDS-PAGE.
³Serum collected from healthy human volunteers was pooled together and diluted 1/500 and immunoblotted.
⁴Mouse sera collected after immunization with a membrane protein-enriched S. pyogenes antigenic preparation was pooled, diluted 1/500, and immunoblotted. These mice were protected against a lethal S. pyogenes challenge.
[0112]
Example 6
This example demonstrates the accessibility of S. pyogenes BVH-P7 protein on the surface of intact streptococcal cells to antibodies.
Bacteria were grown at 37 ° C. in 8% at 37 ° C. in Tood Hewitt (TH) medium (Difco Laboratories, Detroit, MI) with 0.5% yeast extract (Difco Laboratories) and 0.5% peptone extract (Merck, Darmstadt, Germany). CO₂Grown in atmosphere, 0.600 OD₄₉₀nm (-10⁸CFU / ml). A dilution of anti-BVH-P7 or control serum was then added and allowed to bind to cells, which were incubated for 2 hours at 4 ° C. Samples were washed four times with blocking buffer (phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA)) and then 1 ml of goat fluorescein (FITC) conjugated anti-mouse IgG + IgM diluted in blocking buffer was added. did. After an additional 60 minutes incubation at room temperature, the samples were washed four times with blocking buffer and fixed with 0.25% formaldehyde in PBS buffer for 18-24 hours at 4 ° C. Cells were washed twice with PBS buffer and resuspended in 500 μl of PBS buffer. Cells were kept in the dark at 4 ° C. until analyzed by flow cytometry (Epics® XL; Beckman Coulter, Inc.). 10,000 intact S. pyogenes cells per sample were analyzed and the results were expressed as the presence of labeled cells and the fluorescence index. The fluorescence index was calculated as the median fluorescence value obtained after labeling streptococcal cells with the immune serum, divided by the fluorescence value obtained for the control mouse serum. A fluorescence value of 1 indicated the absence of antibody binding on the surface of intact streptococcal cells.
[0113]
Serum collected from eight mice immunized with the BVH-P7 His-tagged recombinant protein was analyzed by cytofluorometry and the results are presented in Table 4. All of the sera collected from mice immunized with the purified BVH-P7 His-tagged protein had their corresponding surface-exposed epitopes on the heterologous (ATCC 12384; serotype M3) S. pyogenes strain tested. BVH-P7 specific antibody that efficiently recognizes The fluorescence index varied from 10 to 18. More than 97% of the 10,000 S. pyogenes cells analyzed were labeled with antibodies present in the BVH-P7 specific antiserum. These sera were also pooled and reacted with the following S. pyogenes strains: serotype M1 S. pyogenes strain ATCC 700294, serotype M3 and serotype M18 S. pyogenes strain ATCC 12357, from the American Type Culture Collection (Rockville, NY). Serotypes M6 S. pyogenes SPY69 and M2 S. pyogenes SPY68 clinical isolates were provided by Centre de recherche en infectiologie du Center hospitalier de 1'universite Laval, Sainte-Foy. BVH-P7-specific antibodies present in pools of serum collected after immunization with purified His-tagged recombinant BVH-P7 protein have a fluorescence index between 4 and 9 The Streptococcus strain adhered to the surface of each bacterium. In contrast, no labeling of streptococcal cells was observed when using pools or serum collected from non-immunized or sham-immunized mice. These observations clearly demonstrate that the BVH-P7 protein is accessible to the surface, where it can be easily recognized by antibodies. Anti-S. Pyogenes antibodies have been shown to play an important role in protection against S. pyogenes infection.
[0114]
Table 4 Evaluation of BVH-P7 specific antibody adhesion on the surface of intact cells of S. pyogenes ATCC 12384 strain (serotype M3)
[Table 5]

¹Mice S1 to S8 were injected subcutaneously three times at 3 weeks intervals with 20 μg of purified BVH-P7 recombinant protein mixed with 10 μg of QuilA adjuvant (Cedarlane Laboratories, Hornby, Canada). The serum was diluted 1/50.
²The fluorescence index was calculated as the median fluorescence value obtained after labeling streptococcal cells with the immune serum divided by the fluorescence value obtained for control mice. A fluorescence value of 1 indicated the absence of antibody binding on the surface of intact streptococcal cells.
³% Of Streptococcus-labeled cells out of 10,000 cells analyzed
⁴Serum collected from non-immunized or mock-immunized mice was pooled 1/50 diluted and used as a negative control for this assay.
20 μg of purified Streptococcal recombinant M protein, a serum obtained from a mouse immunized with a known surface protein, was diluted 1/200 and used as a positive control for the assay.
[0115]
Example 7
This example demonstrates protection against fetal S. pyogenes infection induced by passive immunization of mice with rabbit hyperimmune serum.
New Zealand rabbits (Charles River Laboratories, St-Constant, Canada) were produced and purified subcutaneously as described in Example 4 and BVH-P7 His-tag adsorbed to Alhydrogel adjuvant (Superfos Biosector a / s). Multiple sites were injected at 50 μg and 100 μg of the additional recombinant protein. Rabbits were immunized three times at three week intervals with BVH-P7 His-tagged recombinant protein. Blood samples were collected three weeks after the third injection. Antibodies present in the serum were purified by sedimentation using 40% saturated ammonium sulfate. Groups of 10 female CD-1 mice (Charles River) were immunized intravenously with rabbits immunized with BVH-P7 His-tagged recombinant protein, or with an unrelated control recombinant protein. Injections were made with 500 μl of purified serum collected from rabbits. After 18 hours, the mice were approximately 2 × 10⁷  CFU type 3 was challenged with S. pyogenes strain ATCC 12384. A sample of the S. pyogens challenge inoculum was placed on an agar plate to determine the CFU and confirm the challenge dose. Deaths were recorded for a period of 5 days.
[0116]
Example 8
This example demonstrates the protection of mice against fetal S. pyogenes infection induced by immunization with purified recombinant BVH-P7 protein.
A group of 8 female Balb / c (Charles River, St-Constant, Quebec, Canada) was subcutaneously injected to affinity purified BVH-P7 in the presence of 10 μg QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada). Immunizations were performed three times at two-week intervals with 20 μg of His-tagged recombinant protein or QuilA adjuvant alone in PBS as a control. Blood samples were collected from the orbital sinus two days after the third injection (day 42) on days 1, 14, and 28 before each immunization. One week later, the mice were challenged with approximately 2 × 10 6 CFU of the type 3 S. pyogenes strain ATCC12384. A sample of the S. pyogenes challenge inoculum was placed on a blood agar plate to determine the CFU and confirm the challenge dose. Four out of eight mice immunized with purified recombinant BVH-P7 protein were protected against lethal challenge, compared to only 12% (1/8) of the mice receiving adjuvant alone. (Table 1).
[0117]
Table 5 Ability of recombinant BVH-P7 protein to induce protection against GAS strain ATCC 12384 (type 3)
[Table 6]

Claims (29)

(A) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(B) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(C) a polynucleotide encoding a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(D) a polynucleotide encoding a polypeptide capable of producing an antibody having binding specificity to a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(E) a polynucleotide encoding a portion having a epitope of a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(F) a polynucleotide comprising a sequence selected from SEQ ID NO: 1 or a fragment or analog thereof,
(G) a polynucleotide that is complementary to the polynucleotide of (a), (b), (c), (d), (e) or (f);
An isolated polynucleotide comprising a polynucleotide selected from: (A) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2,
(B) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence selected from SEQ ID NO: 2;
(C) a polynucleotide encoding a polypeptide comprising a sequence selected from SEQ ID NO: 2,
(D) a polynucleotide encoding a polypeptide capable of raising an antibody having binding specificity to a polypeptide comprising the sequence selected from SEQ ID NO: 2,
(E) a polynucleotide encoding a portion having an epitope of a polypeptide comprising a sequence selected from SEQ ID NO: 2,
(F) a polynucleotide comprising a sequence selected from SEQ ID NO: 1,
(G) a polynucleotide that is complementary to the polynucleotide of (a), (b), (c), (d), (e) or (f);
An isolated polynucleotide comprising a polynucleotide selected from: 2. The polynucleotide according to claim 1, wherein said polynucleotide is DNA. 3. The polynucleotide according to claim 2, wherein said polynucleotide is DNA. The polynucleotide according to claim 1, wherein said polynucleotide is RNA. 3. The polynucleotide according to claim 2, wherein said polynucleotide is RNA. Under stringent conditions,
The polynucleotide of claim 1, which hybridizes to either (a) a DNA sequence encoding the polypeptide or (b) the complement of the DNA sequence encoding the polypeptide,
Here, a polynucleotide, wherein the polypeptide comprises SEQ ID NO: 2 or a fragment or analog thereof. Under stringent conditions,
3. The polynucleotide of claim 2, which hybridizes to either (a) a DNA sequence encoding the polypeptide or (b) the complement of the DNA sequence encoding the polypeptide,
Here, a polynucleotide, wherein the polypeptide comprises SEQ ID NO: 2. Under stringent conditions,
The polynucleotide of claim 1, which hybridizes to either (a) a DNA sequence encoding the polypeptide or (b) the complement of the DNA sequence encoding the polypeptide,
A polynucleotide wherein the polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising SEQ ID NO: 2 or a fragment or analog thereof. Under stringent conditions,
3. The polynucleotide of claim 2, which hybridizes to either (a) a DNA sequence encoding the polypeptide or (b) the complement of the DNA sequence encoding the polypeptide,
Wherein the polypeptide comprises at least 10 contiguous amino acid residues from the polypeptide comprising SEQ ID NO: 2. A vector comprising the polynucleotide of claim 1, wherein said DNA is operably linked to an expression control region. A vector comprising the polynucleotide of claim 3, wherein said DNA is operably linked to an expression control region. A host cell transfected with the vector of claim 11. A host cell transfected with the vector of claim 12. 14. A method for producing a polypeptide, comprising culturing the host cell of claim 13 under conditions suitable for expression of said polypeptide. 15. A method for producing a polypeptide, comprising culturing the host cell of claim 14 under conditions suitable for expression of said polypeptide. (A) a polypeptide having at least 70% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2 or a fragment or analog thereof;
(B) a polypeptide having at least 95% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2 or a fragment or analog thereof;
(C) a polypeptide comprising a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(D) a polypeptide capable of producing an antibody having binding specificity to a polypeptide having a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof;
(E) a portion having an epitope of a polypeptide having a sequence selected from SEQ ID NO: 2 or a fragment or analog thereof,
(F) the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminal Met is deleted;
(G) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the secreted amino acid sequence is deleted;
An isolated polypeptide comprising a polypeptide selected from: (A) a polypeptide having at least 70% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2,
(B) a polypeptide having at least 95% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2;
(C) a polypeptide comprising a sequence selected from SEQ ID NO: 2 (d) a polypeptide capable of producing an antibody having binding specificity for a polypeptide having a sequence selected from SEQ ID NO: 2,
(E) a portion having an epitope of a polypeptide having a sequence selected from SEQ ID NO: 2,
(F) the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminal Met is deleted;
(G) the polypeptide of (a), (b), (c), (d), (e) or (f), wherein the secreted amino acid sequence is deleted;
An isolated polypeptide comprising a polypeptide selected from: A chimeric polypeptide comprising two or more polypeptides having a sequence selected from SEQ ID NO: 2 or fragments or analogs thereof, provided that the polypeptides are linked to form a chimeric polypeptide. I do. A chimeric polypeptide comprising two or more polypeptides having a sequence selected from SEQ ID NO: 2, provided that the polypeptides are linked so as to form a chimeric polypeptide. 21. A pharmaceutical composition comprising the polypeptide of any of claims 17 to 20 and a pharmaceutically acceptable carrier, diluent or adjuvant. Erysipelas, pharyngitis and impetigo, scarlet fever, and invasive in hosts susceptible to erysipelas, pharyngitis and impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis and also toxic shock 22. A method for prophylactic or therapeutic treatment of a disease, such as bacteremia and necrotizing fasciitis, comprising administering to the host a prophylactic or therapeutic amount of the composition of claim 21. 22. A method for the prevention or therapeutic treatment of Sterptococcus pyogenes bacterial susceptibility in a host susceptible to Streptococcus pyogenes infection, comprising administering to the host a prophylactic or therapeutic amount of the composition of claim 21. . 23. The method of claim 22, wherein said host is an animal. 23. The method of claim 22, wherein said host is a human. A method for the diagnosis of Streptococcal infection in a host susceptible to Streptococcal infection, comprising:
(A) obtaining a biological sample from a host;
(B) incubating an antibody or fragment thereof reactive with the streptococcal polypeptide of any of claims 17 to 20 with said biological sample to form a mixture;
(C) detecting specifically bound antibodies or bound fragments in the mixture, indicating the presence of streptococcal;
A method that includes A method for the detection of an antibody specific for a Streptococcus antigen in a biological sample, comprising:
(A) obtaining a biological sample from a host;
(B) incubating one or more streptococcal polypeptides of any of claims 17 to 20 with a biological sample to form a mixture;
(C) a method comprising detecting specifically bound antibody or bound fragment in the mixture, which indicates the presence of an antibody specific for Streptococcus. Use of a pharmaceutical composition in the manufacture of a medicament for the prophylactic or therapeutic treatment of a Streptococcus infection. A kit comprising the polypeptide of any one of claims 17 to 20 for detecting or diagnosing a Streptococcal infection.

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